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
[email protected],
[email protected]
Objectives of the combination of direct detection methods • Mass detection based on acoustic sensors (QCM, SAW) → provides layer density ρ and thickness d • Dielectric/optical index variations (impedimetric sensors, optical sensors) → planar multilayer simulations → ellipsometry/SPR/waveguide sensor provides optical index n of layers and thickness d • 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
photodetector
laser beam
Pt CE Cu RE PDMS or SU8+glass
glass prism Pt (CE) (RE)
teflon liquid cell
AFM cantilever
ST quartz substrate
Au WE
viton O−ring
Al IDT glass prism
QCM
1 nF Z (15 MHz) =11 Ω Z (25 MHz) =6 Ω
Q−Sense QCM parameters measurement setup Gamry potentiostat (WE) L=100µ H
reflected laser incoming laser
Z (15 MHz) =9420 Ω Z (15 MHz) =15708 Ω
(670 nm)
AFM/QCM combination setup
SAW/SPR combination setup
QCM/AFM combination 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 roughness) 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 topography (usually only observed for very high concentrations of proteins), individual molecule imaging very difficult on evaporated gold.
∆f3/3 ∆f5/5 ∆f7/7
0
−20
2.3 mg/ml IgG
n
∆f /n (Hz)
−40
−60
−80
−100
500
1000
1500
2000 time (s)
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3500
4000
SPR/SAW combination • Development of Love mode SAW devices with improved sensitivity over QCM and open backside for injection 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 optical index and thickness evolution ⇒ reduce the number of variables to water content and thickness (instead of ρ, n and d). Anti-PSA antibody presented here synthesized at VUB, protocol developed by L. Huang.
55 A EDC/ NHS N7, 186 µg/ml
SAW φ (o)
50
A
E
A
acetate buffer
G G H
H
45
H
PSA 1 µg/ml 1A7, 25 µg/ml
1A7
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5000
6000 7000 time (s)
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9000
10000 11000 12000
2000 SPR angle shift (mo)
E 1900
A
acetate buffer
1800 1700
A
H
A
A 1000
H PSA 100 ng/ml
A=acetate buffer, pH=5.5 E=ethanolamine H=HBS
1600 1500
G
N7, 186 µg/ml
2000
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6000 7000 time (s)
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9000
10000 11000 12000
Experimental setup applied to electrodepsotion of copper
Future improvements nAu(636 nm)=0.1882+i3.07, nAu(670 nm)=0.1511+i3.317, add 5 nm protein (n=1.45)
SOFTWARE CRASH ...
200 0 ∆θ (mo)
2.6
2.4
2% NaOCl
2% NaOCl 100 µg/ml S−layer
−200 100 µg/ml S−layer
−400
buffer
buffer 605 mo
2.2
670 nm+633 nm
−600 buffer
2
−800
500
1000
1500 time (s)
2000
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3000
1.8
1.6
−800
1.4
−1000
o
∆θ (m )
reflected intensity
• All three techniques in one instrument ? • Multiple wavelength SPR by combining lasers using a beam splitter or white light source +diffraction grating ? → issue of interference fringes due to piezoelectric substrate Right: simultaneous measurement of Slayer 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.
1.2
1
669.5 nm 68
70
72
74 θ (o)
−1200 579 mo
−1400
636 nm
−1600 76
78
80
Copy of this poster and related references available at http://mmyotte.free.fr/chua
510 mo
670 nm alone red=(633+670 nm) curve−670 nm curve−1100 ie should be 633 nm only
0
500
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1500 time (s)
2000
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