PHOTONICS RESEARCH GROUP 1

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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 detector

Prism

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

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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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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zoom

avidin 50ng/ml

avidin 10ng/ml

Important when studying kinetics, e.g. drug discovery