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

2

Outline

• Gene Fishing• Immunosensing• Fluid Dynamics

3

Cantilever SensorsStatic mode Dynamic mode

4

Fritz et al, Science 288, 316-318 (2000)

Gene Fishing

5

Thiol modified unlabelled DNA oligomer

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

7

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

9

Immunosensing

10

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)

12N. Backmann

ScFv (100 µg/ml)

Casein (0.5 mg/ml)PEG-silane (2 mM)

Au/Ti (20/2nm)

Functionalization

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.

14

-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

15

µFab by fluid dynamics

• Self-assembling micro- and nanodevices• nanotube chips• LED displays

16

Chladni figures

Sand particles on oscillating plates

Ernst Chladni (1756-1827)

17

Cantilevers in air

500 µm

30 kHz stroboscopic illumination

18

Cantilevers in water& microbeads

f = 300 kHz

Cantilevers:500×100×7 µm3

ferrite

SHSHSHSH

PS

4 µm

19

Chladni figures4th, 5th, 6th Flexural Mode:

20

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

21

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

22

Drag force on beads

0 L

0 L

0 L

0 L

4 µm

4 µm

23

Large & small beads

Merck Estapor, France

4 µm

ferrite

SHSHSHSH

PS

D

Suspended in a surfactant solution

0.5 µm

0.5 µm

24

Large vs. small beads4th mode

4 µm

0.5 µm

Microelectrode arraysMicroelectrode arraysNo No lithographylithographyNanotubeNanotube chipschipsµµLED displaysLED displays

25

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

26

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

27

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

28

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

29

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%

31

Edge vortices at antinodes

2nd mode

85 kHz

70 kHz

32

Simple model

viscous non-linear inertia

linear inertia

pressuregradient

Navier-Stokes:

Simple Navier-Stokes:

33

Simple model

34

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)

35

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

36

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

37

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)

38

Mass Sensing

39

40

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

41

Static vs. Dynamic Mode

Convert .mov to .wmv

42

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)

43

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)

44

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)

45

Beads in vortices:simulation

Petros Koumoutsakos and Jens Walther, ETH Zurich

46

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

47

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

48

Edge Vortices 2nd mode:80 kHz

49

Edge vortices

node(2nd mode)