Introduction to biosensors

1. Introduction to biosensors Peter Bienstman

2. Biosensors • Detect presence and concentration of biomolecules • DNA • Proteins • Virus • Bacteria • … • Two classes: • Labeled: indirect detection • Label-free: direct detection

3. Applications • Diagnostics • Drug development • Food safety • Environmental monitoring • …

4. Desired characteristics • Low limit of detection (“sensitivity”) • Selective • Reproducible • Cheap • Portable • Fast • Multi-parameter • …

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!

6. elisa

7. Elisa tests • Enzyme-LinkedImmunoSorbentAssay • Workhorse of protein detection • Detect protein by using • fluorescent labels • labels with enzymes that start a colouring reaction on a dye substrate • …

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)

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

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

11. Test result

12. Au nanoparticles

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)

14. Au nanoparticles Immobilised on latex beads Two different particles sizes In solution

15. Ways to use them • As a fancy dye • Changing colour on aggregation • Combined with latex beads • …

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

17. Changing colour on aggregation Colloidal gold coated with hCG antibody

18. Changing colour on aggregation hCG present

19. Changing colour on aggregation Absorption band shifts due to aggregation and colourchanges (see later)

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

21. Quantum dot labels

22. Quantum dot labels • Alternative to metallic nanoparticles • Typically colloidally grown • PbSe, CdTe, … • Much sharper spectra, widely tuneable by size

23. Bead based assays

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

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

26. Read-out in flow cytometer • E.g., one laser measures label on bead, the other measures the reporter fluorophore

27. Colour-encoded beads e.g. LuminexxMAP technology, 2 fluorescent dyes in different ratios

28. Labelfree sensors

29. Labeling • detect a molecule by attaching a label to it • very sensitive (10-9...10-16mol/l) • commercial product (Elisa, DNA arrays, ..)

30. Disadvantages to labeling?

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

32. Label-free sensors • detect presence of biomolecules directly • focus here: label-free optical biosensors • selective binding causes refractive index change flow with biomolecules matching biomolecule (analyte) biorecognition element (ligand)

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, …)

34. Surface plasmon resonance sensor

35. Plasmons • Collective wave oscillations of electrons in a metal motion of electrons propagation of wave Fig: R. Nave, Hyperphysics

36. Surface plasmons • Interaction between: • plasmon at surface of metal • electromagnetic wave EM wave plasmon

37. Magnitude of EM field Cannot be excited directly from the outside position light intensity

38. angle Reflection experiment reflection angle

39. Towards a biosensor reflection angle angle

40. R  From source To detector Prism Gold 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

41. advantages • very sensitive, index differences of 10-6 possible • functionalised Au layers off-the-shelf available • integrated microfluidics • but • bulky • expensive • difficult to integrate and multiplex

42. Evanescent wave sensors

43. Evanescent wave biosensor Densmore, 2008

44. 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

45. Effective index change still needs to be translated into something measurable. • Many possibilities: • Resonators • Interferometers • …

46. Evanescent wave sensors: resonators

47. Ring resonators 1.55 μm Binding of biomoleculeschange of refractive index  resonance wavelength shift

48. Towards a better sensor • High demandsonread-out system, but filters noise Larger shift Narrower dips More interactionbetweenlight and molecules

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 Δλmin : smallest distinguishablewavelength shift

50. 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!