The metrology of biosensors: a multiparameter approach to characterizing protein layers
J.-M. Friedt, L. Francis, K.-H Choi, A. Campitelli IMEC, Kapeldreef 75, 3001 Leuven, Belgium
friedtj@imec.be, campi@imec.be
Objectives of the combination of direct detection methods
• Massdetection based on acoustic sensors (QCM, SAW)
→provides layer densityρand thickness d
• Dielectric/optical index variations (impedimetric sen- sors, optical sensors) → planar multilayer simulations → ellipsometry/SPR/waveguide sensor provides optical index nof layers and thicknessd
• scanning probe microscopies → surface morphology, chemical properties when using functionalized tips
⇒ combine these methods to obtain independent estimates of layer thickness and water content of the protein layers
(15 MHz)
Z =9420Ω
viton O−ring teflon liquid cell
QCM AFM cantilever
glass prism laser beam photodetector
measurement setup Gamry potentiostat
(WE) Q−Sense QCM parameters
L=100 Hµ
(25 MHz)
(15 MHz)
Z =6
Ω Z =15708 Pt (CE)
(RE)
1 nF
(15 MHz)
Z =11Ω Ω
Cu RE Pt CE
PDMS or
ST quartz substrate SU8+glass
(670 nm) incoming laser
laser reflected glass prism Al IDT
Au WE
AFM/QCM combination setup SAW/SPR combination setup
QCM/AFM combination
500 1000 1500 2000 2500 3000 3500 4000
−100
−80
−60
−40
−20 0
time (s)
∆f n
/n (Hz)
∆f3/3
∆f5/5
∆f7/7
2.3 mg/ml IgG
Use of commercial instruments:
• QSense-AB QCM monitoring electronics (frequency overtones and damping)
→continuous monitoring of the 3rd, 5th and 7th overtones+quality factor
• Molecular Imaging AFM (moving scanner, fixed sample holder)
• Gamry potentiostat for electrochemistry applications
Problems of viscous interactions, trapped water, QCM/AFM interaction (oscillation amplitude: '3 nm ; standing wave pattern disturbs QCM resonance frequency).
Application to electrochemistry (relate QCM behavior to electrodeposited film rough- ness) and to biology (example presented here: IgG adsorption on hydrophobic-thiol coated gold (data obtained by Z. Cheng).
Problem: bare AFM tips provide little information on the surface other than to- pography (usually only observed for very high concentrations of proteins), individual molecule imaging very difficult on evaporated gold.
SPR/SAW combination
1000 2000 3000 4000 5000 6000 7000 8000 9000 100001100012000 40
45 50 55
time (s) SAW φ (o)
1000 2000 3000 4000 5000 6000 7000 8000 9000 100001100012000 1500
1600 1700 1800 1900 2000
time (s) SPR angle shift (mo)
A A
A=acetate buffer, pH=5.5 N7,
186 µg/ml A
E
E=ethanolamine acetate buffer G
A H H PSA 100 ng/ml
H=HBS A N7, 186 µg/ml EDC/
NHS A
E acetate buffer
G A
PSA 1 µg/ml
1A7, 25 µg/ml H
H G
1A7 H
• Development of Love mode SAW devices with im- proved sensitivity over QCM and open backside for in- jection of laser
•modified Ibis II SPR instrument
→ limited effect of viscous interactions but problem with birefringence of piezoelectric substrate⇒separate SPR dip from interference fringes minima
→single wavelength SPR leads to uncertainty on opti- cal index and thickness evolution⇒reduce the number of variables to water content and thickness (instead of ρ,nandd). Anti-PSA antibody presented here synthe- sized at VUB, protocol developed by L. Huang.
Experimental setup applied to elec- trodepsotion of copper
Future improvements
•All three techniques in one instrument ?
• Multiple wavelength SPR by combin- ing lasers using a beam splitter or white light source +diffraction grating ? →issue of interference fringes due to piezoelectric substrate
Right: simultaneous measurement of S- layer proteins adsorption on gold coated glass monitored at 633 and 670 nm, and related simulations showing that the angle
shift is dependent on the wavelength. 1 68 70 72 74 76 78 80
1.2 1.4 1.6 1.8 2 2.2 2.4 2.6
reflected intensity
θ (o)
nAu(636 nm)=0.1882+i3.07, nAu(670 nm)=0.1511+i3.317, add 5 nm protein (n=1.45)
669.5 nm 636 nm
500 1000 1500 2000 2500 3000
−800
−600
−400
−200 0 200
time (s)
∆θ (mo)
0 500 1000 1500 2000 2500 3000
−1600
−1400
−1200
−1000
−800
time (s)
∆θ (mo) buffer
100 µg/ml S−layer
100 µg/ml S−layer
2% NaOCl 2% NaOCl
buffer buffer
670 nm alone 670 nm+633 nm
red=(633+670 nm) curve−670 nm curve−1100 ie should be 633 nm only 605 mo
510 mo 579 mo
SOFTWARE CRASH ...
Copy of this poster and related references available athttp://mmyotte.free.fr/chua
