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

P HOTONICS R ESEARCH G ROUP 2 Biosensors Detect presence and concentration of biomolecules DNA Proteins Virus Bacteria … Two classes: Labeled: indirect detection Label-free: direct detection

P HOTONICS R ESEARCH G ROUP 3 Applications Diagnostics Drug development Food safety Environmental monitoring …

P HOTONICS R ESEARCH G ROUP 4 Desired characteristics Low limit of detection (“sensitivity”) Selective Reproducible Cheap Portable Fast Multi-parameter …

P HOTONICS R ESEARCH G ROUP 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!

P HOTONICS R ESEARCH G ROUP 6 ELISA

P HOTONICS R ESEARCH G ROUP 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 …

P HOTONICS R ESEARCH G ROUP 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)

P HOTONICS R ESEARCH G ROUP 9 Test principle See animations at

P HOTONICS R ESEARCH G ROUP 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

P HOTONICS R ESEARCH G ROUP 11 Test result

P HOTONICS R ESEARCH G ROUP 12 AU NANOPARTICLES

P HOTONICS R ESEARCH G ROUP 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)

P HOTONICS R ESEARCH G ROUP 14 Au nanoparticles Two different particles sizes In solution Immobilised on latex beads

P HOTONICS R ESEARCH G ROUP 15 Ways to use them As a fancy dye Changing colour on aggregation Combined with latex beads …

P HOTONICS R ESEARCH G ROUP 16 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

P HOTONICS R ESEARCH G ROUP 17 Colloidal gold coated with hCG antibody Changing colour on aggregation

P HOTONICS R ESEARCH G ROUP 18 hCG present Changing colour on aggregation

P HOTONICS R ESEARCH G ROUP 19 Absorption band shifts due to aggregation and colour changes (see later) Changing colour on aggregation

P HOTONICS R ESEARCH G ROUP 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

P HOTONICS R ESEARCH G ROUP 21 QUANTUM DOT LABELS

P HOTONICS R ESEARCH G ROUP 22 Quantum dot labels Alternative to metallic nanoparticles Typically colloidally grown PbSe, CdTe, … Much sharper spectra, widely tuneable by size

P HOTONICS R ESEARCH G ROUP 23 BEAD BASED ASSAYS

P HOTONICS R ESEARCH G ROUP 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

P HOTONICS R ESEARCH G ROUP 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

P HOTONICS R ESEARCH G ROUP 26 Read-out in flow cytometer E.g., one laser measures label on bead, the other measures the reporter fluorophore

P HOTONICS R ESEARCH G ROUP 27 Colour-encoded beads e.g. Luminex xMAP technology, 2 fluorescent dyes in different ratios

P HOTONICS R ESEARCH G ROUP 28 LABELFREE SENSORS

P HOTONICS R ESEARCH G ROUP 29 Labeling detect a molecule by attaching a label to it very sensitive ( mol/l) commercial product (Elisa, DNA arrays,..)

P HOTONICS R ESEARCH G ROUP 30 Disadvantages to labeling?

P HOTONICS R ESEARCH G ROUP 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

P HOTONICS R ESEARCH G ROUP 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

P HOTONICS R ESEARCH G ROUP 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, …)

P HOTONICS R ESEARCH G ROUP 34 SURFACE PLASMON RESONANCE SENSOR

P HOTONICS R ESEARCH G ROUP 35 Plasmons Collective wave oscillations of electrons in a metal Fig: R. Nave, Hyperphysics motion of electrons propagation of wave

P HOTONICS R ESEARCH G ROUP 36 Surface plasmons Interaction between: plasmon at surface of metal electromagnetic wave EM wave plasmon

P HOTONICS R ESEARCH G ROUP 37 Magnitude of EM field light intensity position Cannot be excited directly from the outside

P HOTONICS R ESEARCH G ROUP 38 Reflection experiment reflection angle

P HOTONICS R ESEARCH G ROUP 39 Towards a biosensor reflection angle

P HOTONICS R ESEARCH G ROUP 40 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 Gold R

P HOTONICS R ESEARCH G ROUP 41 advantages o very sensitive, index differences of possible o functionalised Au layers off-the-shelf available o integrated microfluidics but o bulky o expensive o difficult to integrate and multiplex

P HOTONICS R ESEARCH G ROUP 42 EVANESCENT WAVE SENSORS

P HOTONICS R ESEARCH G ROUP 43 Evanescent wave biosensor Densmore, 2008

P HOTONICS R ESEARCH G ROUP 44 Influence of mode profile profile should overlap maximally with the adlayer, and not with bulk fluid (noise!) high index contrast is best Low contrastHigh contrast

P HOTONICS R ESEARCH G ROUP 45 Effective index change still needs to be translated into something measurable. Many possibilities: Resonators Interferometers …

P HOTONICS R ESEARCH G ROUP 46 EVANESCENT WAVE SENSORS: RESONATORS

P HOTONICS R ESEARCH G ROUP 47 Ring resonators Binding of biomolecules  change of refractive index  resonance wavelength shift 1.55 μm

P HOTONICS R ESEARCH G ROUP 48 Towards a better sensor High demands on read-out system, but filters noise More interaction between light and molecules Narrower dips Larger shift

P HOTONICS R ESEARCH G ROUP 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 Δλ min : smallest distinguishable wavelength shift

P HOTONICS R ESEARCH G ROUP 50 What determines Δλ min ? 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 Δλ min can get smaller than measurement resolution!

P HOTONICS R ESEARCH G ROUP 51 Example: measurement setup

P HOTONICS R ESEARCH G ROUP 52 Surface sensing: biotin/avidin High avidin concentrations: saturation Low avidin concentrations: quantitative measurements Detection limit: lower than 3ng/ml

P HOTONICS R ESEARCH G ROUP 53 Real time measurement zoom avidin 50ng/ml avidin 10ng/ml Important when studying kinetics, e.g. drug discovery