1. Class 5 Immuno-assay with magnetic nanoparticle tags Gaster et al Nature Medicine 15:1327 (2009) Basic idea Giant magneto resistance (GMR) Sensor characterization

2. Sandwich immunoassay with superparamagnetic particle tags and GMR detection of magnetic labels

3. Reaction well: tygon tubing glued to GMR sensor surface 5mm 3mm well vol  125  l sample vol 50  l sample height  1.5mm

4. What is GMR*? electrons interact differently with mag. materials depending on alignment of their spin with local magnetic field – more scattering (more resistance) when spin and field are parallel *used in hard drive read heads, Nobel prize in physics 2007

5. GMR “spin valve” sensor 17nm 40nm Apply H = H y sin  t j then R oscillates with freq.  (or 2  ) +-

6. 50nm Applied H a Fe Superparamagnetic: mag moments not aligned (at RT) in absence of ext. field Mag field from beads (if present) adds to GMR resistance

7. How far from bead is its magnetic field felt? dipole field  (a/r) 3, so  100nm for  50nm beads How wide is sensor strip? How should bead effect on resistance vary with # beads?

8. Previous expt used 16nm beads on 200nm-wide sensors Change in resistance was  number of beads stuck

9. Signal processing They apply current I, measure V (=IR) as surrogate for R They apply oscillating H(  1 ) -> oscillating R(  1 ), V(  1 ) They also vary I(  2 ) -> V varying with  1,  2,  1 +  2 Measure rms amplitude of  V(  1 +  2 ) Repeat after adding allowing beads to attach i.e. measure  V) due to beads

10. Other things to worry about Temperature effects T may change due to environmental drift or joule heating with current thru sensor T alters bulk resistance  V BR =  T T also alters GMR effect  V GMR (  ) =  T They know , use  V(  =0) to est. local  T, then correct  V(  1 +  2 ) for temp effect on GMR Caveat: the magnitude of temperature effects are comparable to changes in V from beads

11. How do GMR sensor results differ from ELISA? Does GMR extension to right => not all receptors bind analyte at 500pM? If all bound at 5nM, what fraction should be bound at 5fM? How may receptors are bound at 5fM? Why is  V  [c 0 ] 1/3 ?

12. Aside on fraction of receptors that bind analyte and fraction of analyte that binds receptors ar/r 0 = a 0 /(K D +a 0 +r 0 ) [1+ a 0 r 0 /(K D +a 0 +r 0 +) 2 + … ] ar/a 0 = r 0 /(K D +a 0 +r 0 ) [1+ a 0 r 0 /(K D +a 0 +r 0 +) 2 + … ] a 0 = initial conc of analyte (per sample vol) r 0 = initial conc of receptors (per sample vol) Useful when correction term in power series is small and sample volume is fixed (i.e. no flow) See spread sheet!

13. Why is limit of detection for CEA 25X lower than for lactoferrin?

14. Time course of  V Why so fast? Suppose conc. of beads is 4nM, K D of biotin-SA bond is 10 -15 M, and k on is the usual 10 6 /Ms In this case c 0 /K D >>1 so  rxn = (1/k off )/(1+c 0 /K D ) simplifies to  1/(c 0 k on )

15. Amplification = after beads bind, add any protein w/several biotins, then more SA-magnetic nanoparticles Can they detect 50aM analyte? If so, how many analyte molecules should be bound?

16. Shows great reproducibility in different conditions, but are results for CEA the same as in Figure 2?

17. GMR immunoassay for serum CEA assay can be used to monitor growth of tumor that secretes CEA in mice How big a tumor would this correspond to in humans, assuming mice and men are proportional in all relevant ways, the 21 day mouse tumor is 1 g, and mice weigh 30g versus 60kg for men? Might we need more sensitivity?

18. Conclusions 1.GMR immuno-assay has much larger dynamic range than ELISA; this could be very important in multiplex assays for proteins with vastly different concentrations in same sample 2.GMR assay is sensitive, but maybe less than claimed 3.How costly are sensor chips? 4.How costly is sensor? 5.How reliable is data processing, e.g. for temp. correction?

19. Next week: immuno-assay with single-molecule sensitivity based on fluorescence labels and Total Internal Reflection Fluorescence Microscopy (TIRFM) Read Jain et al Nature 473:484 (2011) Basic idea – capture analyte on transparent surface introduce fluorescent label (e.g. on second ab) record fluorescent image in microscope sample negative control

20. TIRF microscopy reduces background, allowing detection of single fluorescent molecules Jargon protein names: YFP, PKA, ADAP, mTor, etc. epitopes (small chemical features, can be peptides, that antibodies bind to): FLAG, HA fluorescent proteins (e.g. from jellyfish, corals): often named for emission color yellow (YFP), red (mCherry) IP = immunoprecipitation, here usually means capture of analyte on surface by antibody FRET – Fluorescence Resonance Energy Transfer: when different fluors are within nm of each other, excited state can transfer -> altered em. color photobleaching – light-induced chem. change killing fluor.

21. Authors describe technique mainly for research purposes: e.g. to detect what other proteins a test protein binds to, or how many molecules in a complex Our focus: how does this method compare to others as a sensor Issues to think about as you read: background, dynamic range, field of view, potential for automation, cost