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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