1
A Novel Diagnostic Assay Based on Nanomechanics
NCCR Nanoscale Science
NASA INAC Molecular Conductivity and Sensor Workshop
West Lafayette
July 27-29, 2005Marko Dorrestijn
6
R. McKendry et al., Proc. Nat. Acad. Sci. USA 99 (2002) 9783-9788
Reference cantilevers
Unspecific background
Overhangs at 3’ and 5’ ends
Concentration dependence
target
DNA cantilever sensor array
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Gene FishingGene Fishing
Complete human genomeComplete human genome
AldolaseAldolase genegene
Housekeeping geneHousekeeping genecopy number ~ 6000copy number ~ 6000
Three 22Three 22--24 24 bpbp sequences within sequences within the background of 30 million the background of 30 million basepairsbasepairs
Three cantilevers: Three cantilevers: aldolasealdolase sequencessequencesTwo cantilevers: reference sequencesTwo cantilevers: reference sequences
80 5 10 15 20 25 30 35 40 45 50 55
-40
-20
0
20
40
60
Diff
eren
tial d
efle
ctio
n (n
m)
Time (min)
Aldolase – 24bp unsp (1)BioB1 – 24bp unsp (1)24bp unsp (1) – 24 unsp (2)
No labelsNo labelsNo PCR amplificationNo PCR amplification
SpecificitySpecificitySensitivitySensitivity
Fishing an unlabeled gene in total RNA
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Cardiovascular Application• Fast bedside (mobile) diagnostics for intensive care patients • Biomarkers: Creatin kinase, troponin and myoglobin
Patrick Hunziker, University Hospital BaselY. Arntz et al., Nanotechnology 14 (2003) 86-90
11scFv cantilevers
Complete Antibody ideal Complete Antibody realLoss of receptors > 50 %
Fab cantileversNatalija Backmann et al., submitted.
Improving sensitivity of antibody coated cantilevers (high nM -> pM)
13
Injected antigen[1 µM]
Negative controlscFv fragment
Raw data
Differential signal~ 50 nm
scFv antibody fragment activated array
110 115 120 125 130 135 140
0
25
50
75
100
Binding of GCN4-pD to C11L34S-scFv, G9-scFv and BSA
Def
lect
ion
(n
m)
Time (min)
C11L34S G9 BSA
In collaboration withUni Zürich, Plückthun Group
N. Backm
ann
N. Backmann, et al., submitted.
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-100
-50
0
50
100
150
200
250
300
Dif
fere
nti
al s
ign
al (
RU
)
0 20 40 60 80Time, min
Antigen Buffer
15 nM
300 nM
Surface Plasmon Resonance BIAcoreBinding of AR-GCN4 to C11L34cys and G9cysimmobilized on Au sensor chip(differential signal = RC11L34cys-RG9cys)
Cantilever Sensor Array
Binding of AR-GCN4 to C11L34cys
Buffer: HBST
No labelsNo labelsAs sensitive as SPRAs sensitive as SPR
Integrated arrayIntegrated array
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Acoustic Streaming
10 µm/s
10 mm/s
T. Açikalin, A. Raman, S. V. Garimella, JASA 114, 1785 (2003).Collabation with Arvind Raman
4th mode
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Chladni Figures
T. Açikalin, A. Raman, S. V. Garimella, JASA 114, 1785 (2003).
Stream function ? (x,y)
Collabation with Arvind Raman
? ,x = - vy? ,y = vx
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Large & small beads
Merck Estapor, France
4 µm
ferrite
SHSHSHSH
PS
D
Suspended in a surfactant solution
0.5 µm
0.5 µm
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Large vs. small beads4th mode
4 µm
0.5 µm
Microelectrode arraysMicroelectrode arraysNo No lithographylithographyNanotubeNanotube chipschipsµµLED displaysLED displays
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Cantilever sensorsGenomics• No labeling• Repeatable using denaturation / unbinding agents• Physiological sensitivity established, detection of single gene fragments
within complete genomic background
Proteomics• Direct observation of proteins relevant to cardiovascular diseases• Detection of multiple proteins in presence of unspecific background for fast
bedside diagnostics• Increase of sensitivity by factor of 100 using single chain (scFv) fragments.
Compatible with silicon technology• parallelization into integrated devices, DNA chips, binding assays• scalability with microfluidics towards lab-on-a-chip
Fluid dynamics• Regular arrays of micro- and nanobeads• Fabrication of nanoscale electrode arrays for chips based on molecular
electronics
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AcknowledgementUni BaselChristoph GerberErnst MeyerHans Peter LangMartin HegnerFrancois HuberNatalija BackmannThomas Braun Yuri ArntzMurali GhatkesarViola BarwichJiayun ZhangAdriaan BredekampJean-Pierre Ramseyer
IBM ResearchAlexander BietschMichel DespontUte DrechslerPaul SeidlerRolf Allenspach
University Hospital BaselPatrick Hunziker
Roche:Ulrich Certa
Purdue UniversityArvind RamanTolga Açikalin
NCCR Nanoscale Science
Uni ZürichA. Plückthun
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Sample preparation for gene fishing
chromosomal DNA mRNAFrom about
35000 genes
cRNA 100 bases
long fragments
cDNA
Extraction of mRNAfrom cells
Reverse trans-cription of mRNA Detection with probe oligos
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A-l: low GC: ( 22-mer sequences)SH-5' ATTGGAAAATTTCTGGGTGCAA 3‘
A-m: mediumGC: ( 24-mer sequences)SH-5'TCAGGCTCCACAATGGGTACAATG 3‘
A-h: high GC: ( 24-mer sequences)SH-5' GTGACCCCAGGGACGGCAGGGGGC 3‘
poly-T: ( 22-mer sequences)SH-5' TTTTTTTTTTTTTTTTTTTTTTTT 3’
BioB1:SH-5‘ ACA TTG TCG GAA 3‘
BioB2:SH-5‘ TGC TGT TTG AAG 3‘
Unsp: unspecific oligoSH-5‘ACA CAC ACA CAC 3‘
Target gene Reference sequences
Functionalization of each cantilever with a different probe sequence and reference oligo
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Multiple protein detection
Myoglobin 50 µg/mlCreatine Kinase 50 µg/mlBackground 100 µg/ml BSA
-80
-60
-40
-20
0
20
40
Dif
fere
nti
al D
efle
ctio
n [
nm
]
0 5000 10000 15000
Time [s]
M BB C
M Myoglobin injection
C Creatin Kinase injection
B Buffer injection
Patrick Hunziker, University Hospital BaselY. Arntz et al., Nanotechnology 14 (2003) 86-90
30N. Backmann
SPR measurements
Binding of AR-GCN4 After regeneration withGlycine buffer (pH 2.8)
Rmax~50%
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Simple model
viscous non-linear inertia
linear inertia
pressuregradient
Navier-Stokes:
Simple Navier-Stokes:
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Cantilever amplitudes in water
10 100 1000
1
10
100
6
54
3
1 2
Microcantilever (MC) amplitudes in 100µL water per V
pp of piezo drive (peak-peak voltage)
MC: 564x100x7.14µm3 (SEM), siliconDark green data taken at elevated temperature.Array F11 was cleaned in piranha, E10 was not.Assumptions:- amplitude varies linearly w/ drive voltage- node positions in water equal those in vacuum- zero curvature of MC beyond last node
Am
plitu
de (
nm p
er V
pp)
f /kHz
F11 28oC 07.10.03 14:47-16:32 F11 35oC 07.10.03 14:47-16:32 F11 28oC 21-10-03 16:18 F11 27oC 04-11-03 10:51-11:01 E10 25oC 25-02-04 10:29
~1/f1.5 (vacuum: ~1/f 2)
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Chladni Figures
0 200 400 600 800 1000 1200 1400
1
10
100
6
54
3
1 2
Microcantilever (MC) amplitudes in 100µL water per V
pp of piezo drive (peak-peak voltage)
MC: 564x100x7.14µm3 (SEM), siliconDark green data taken at elevated temperature.Array F11 was cleaned in piranha, E10 was not.Assumptions:- amplitude varies linearly w/ drive voltage- node positions in water equal those in vacuum- zero curvature of MC beyond last node
MC
am
plitu
de /n
m p
er V
pp
f /kHz
F11 28oC 07.10.03 14:47-16:32 F11 35oC 07.10.03 14:47-16:32 F11 28oC 21-10-03 16:18 F11 27oC 04-11-03 10:51-11:01 E10 25oC 25-02-04 10:29
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Type of RNA FUNCTION
mRNAs
rRNAs
tRNAs
snRNAs
snoRNAs
Other non-coding RNAs
Messenger RNAs, code for proteins
Ribosomal RNAs, form the basic structureof the ribosome and catalyze protein synthesis
Transfer RNAs, central to protein synthesis asadaptors between mRNA and amino acids
Small nuclear RNAs, function in a variety of nuclear processes, including the splicing ofpre-mRNA
Small nucleolar RNAs, used to process and chemically modify tRNAs
Function in diverse cellular processes, includingtelomere synthesis, X-chromosome inactivation,and the transport of proteins into the ER
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ImmunosensingELISA
peptide antigen = immobilized (biotin-avidin)added 50 nM scFv (w/ his-tag), then anti-his, then anti-anti-his bound to alkaline-phosphatase, then its substrate added (resulting in yellow or green color, dpt on substrate)(d. grey = excess of free peptide)
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INF-α induced gene expression (IFITM1)
Inf-α present (+) Inf-α absent (-)
Injection of RNA of Inf-α treated cells
Injection of RNA ofnot Inf-α treated cells
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Acoustic streaming
1
2
3
4
5
6
0 5 10 15 20 25 30Velocity (µm/s or mm/s)
Mod
e Streaming velocity (µm/s) Cantilever velocity (mm/s)
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Drag force on beads
0 5 10 15-0.04
0.00
m1 m2 m3 m4 m5 m6
-2 0 2 4 6 8 10 12 14 16
-0.8
-0.4
0.0
0.4
F x/p
N
Diameter bead /µm
m1m2m3m4m5m6
(adapted Stokes)
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Critical bead diametervs. frequency
0 250 500 750 1000 1250 15000
2
4
6
8
10
12
14
16
2.8 δ (where δ = (2ν/ω)1/2)
Dia
met
er o
f zer
o ve
loci
ty /µ
m
f /kHz
Diameter of zero velocity (squares)scales with thickness of boundary layer (line)
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Invisible area under MC
N.A. = n sin(a)0.3 = 1.33 sin(a)2 sin(a) = 0.45
2a
cantilever
N.A. = 0.3
n = 1.33
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Bangs Labs beads
Fe3O4
COOHCOOHCOOH
COOH
polystyrene
D = 0.9 µm, Dragon Green fluorescent (480,520)D = 2.8 µm, Flash Red fluorescent (660, 690)
Suspended in a surfactant solution.Density: 1.1 - 1.2 g/cm3
Iron oxide content: 4 - 12 %
D
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