P HOTONICS R ESEARCH G ROUP 1 Introduction to biosensors Peter Bienstman

PHOTONICS RESEARCH GROUP 1

PHOTONICS RESEARCH GROUP

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

Environmental monitoring

…


Desired characteristics

Low limit of detection (“sensitivity”)

Selective

Reproducible

Cheap

Portable

Fast

Multi-parameter

…


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!


ELISA


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• …


Example: pregnancy test

Detects hCG protein (human Chorionic Gonadotropin) in urine

Based on strip which pulls fluid through by capillary action (lateral flow immunochromatography)


Test principle

See animations at http://www.whfreeman.com/kuby/content/anm/kb07an01.htm


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


Test result


AU NANOPARTICLES


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)


Au nanoparticles

Two different particles sizesIn solution

Immobilised on latex beads


Ways to use them

As a fancy dye

Changing colour on aggregation

Combined with latex beads

…


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


Colloidal gold coated with hCG antibody



hCG present



Absorption band shifts due to aggregation and colour changes (see later)



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


QUANTUM DOT LABELS


Quantum dot labels

Alternative to metallic nanoparticles

Typically colloidally grown

PbSe, CdTe, …

Much sharper spectra, widely tuneable by size


BEAD BASED ASSAYS


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


Labeled microcarriers

• Don’t flow fluid over planar substrate, but break up substrate into microcarriers which float in the fluid

• Better mixing properties too


Read-out in flow cytometer

E.g., one laser measures label on bead, the other measures the reporter fluorophore


Colour-encoded beads

e.g. Luminex xMAP technology, 2 fluorescent dyes in different ratios


LABELFREE SENSORS


Labeling

• detect a molecule by attaching a label to it

• very sensitive (10-9...10-16 mol/l)

• commercial product (Elisa, DNA arrays, ..)


Disadvantages to labeling?


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


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


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, …)


SURFACE PLASMON RESONANCE SENSOR


Plasmons

Collective wave oscillations of electrons in a metal

Fig: R. Nave, Hyperphysics

motion of electrons

propagation of wave


Surface plasmons

Interaction between:plasmon at surface of metalelectromagnetic wave

EM wave

plasmon


Magnitude of EM field

light intensity

position Cannot be excited directly from the outside


Reflection experiment

reflection

angle angle


Towards a biosensor

reflection

angleangle


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


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


EVANESCENT WAVE SENSORS


Evanescent wave biosensor

Densmore, 2008


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


Effective index change still needs to be translated into something measurable.

Many possibilities:

• Resonators

• Interferometers

• …


EVANESCENT WAVE SENSORS: RESONATORS


Ring resonators

Binding of biomolecules change of refractive index

resonance wavelength shift

P

P

1.55 μm


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


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


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!


Example: measurement setup


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


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

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P HOTONICS R ESEARCH G ROUP 1 Introduction to biosensors Peter Bienstman