quantitative nanomechanical diagnostics - src

innovating Nanoscience

Quantitative Nanomechanical Diagnostics• Towards portable nanomechanical diagnostic devicesProf. Martin Hegner (hegnerm@tcd.ie)CRANN, School of Physics, Trinity College Dublin, Ireland

© CRANN www.crann.tcd.ie

Goals

• Provide overview on state of the art label free diagnostic.

• Nanomechanical Principle• Genomics• Proteomics• Microbi-omics

© CRANN www.crann.tcd.ie

Diagnostic in Micron – / Nano-Regime

Nanoscience delivers new opportunities for better understanding of those disease related compartments and offers new tools to improve drug treatment. Especially in the context of personalized healthcare we have to understand and be able to analyze low level structures such as supramolecular assemblies

Personalized Health Care

TomorrowNew tests based on

genomics and proteomicsRisk prediction – what could happen?Early detection – what is happening now?Pharmacogenomics – which therapy to use?Therapy prediction – what will happen?

MonitoringTherapyDiagnosisPreventionTarget

MonitoringPredisposition

Testing

test test test test testtest/drug

© CRANN www.crann.tcd.ie

Nanomechanics: Exploring New Frontiers in Biosensing and Diagnostics

Nanometer Scale Force measuring devices allow detailed investigations of energy, kinetics and mechanics of interacting biological molecules.

Cantilever Arrays

IndividualFunctionalization

Device / Assay development

© CRANN www.crann.tcd.ie

Static or dynamic analysis

Qualitative measurementLigand-Receptor InteractionsSurface Stress changes

Quantitative measurementLigand Receptor InteractionsIn-/Decrease of mass

In gas, humid environment, in liquids

Combination of both measurements in one instrument

Static (DC) Dynamic (AC)

•Fritz J. et al.Science (2000)•McKendry R. et al. PNAS(2002)•Arntz Y. et al. Nanotechnology (2003)

•Gfeller, K.Y. et al.Appl. Env. Microbio.(2005) •Nugaeva, N. et al.Microscopy & Microanal.(2007)

•Huber F. et al Biosens.Bioelectr. (2005)•Backmann N. et al. PNAS (2005)•Zhang J. et al. Nature Nanotech.(2006)

Braun et al.,Phys. Rev.E(2005)Braun,T. et al.Nature Nanotech(2009)

© CRANN www.crann.tcd.ie

Functionalization of sensors Assembly of chamber for measurement

M. W

alth

er /

CR

AN

N

© CRANN www.crann.tcd.ie

Genomic studies using cantilever arraysRohit Mishra; Collaboration with Hoffmann-La-Roche (Certa, Noy)

dsRNAViral RNA/Transposons

Nucleus

Cytoplasm

Dicer

RISCComplementary binding

with mRNA

siRNA formationStrand separation

mRNA cleavageSelective Gene

Silencing

mRNAGene Expression

Synthetic siRNADelivery through cell membrane for Therapeutic Gene Silencing

Relevant biomarkers in diagnostics

Si-CL arraysIBM labs

500µm long100µm wide0.5µm thickpitch 250µm

Nature Nanotech. 1 (2006) 214.

Genomics

© CRANN www.crann.tcd.ie

Genomic studies using cantilever arraysRohit Mishra; Collaboration with Hoffmann-La-Roche (Certa, Noy)

dsRNAViral RNA/Transposons

Nucleus

Cytoplasm

Dicer

RISCComplementary binding

with mRNA

siRNA formationStrand separation

mRNA cleavageSelective Gene

Silencing

mRNAGene Expression

Synthetic siRNADelivery through cell membrane for Therapeutic Gene Silencing

Relevant biomarkers in diagnostics

Si-CL arraysIBM labs

500µm long100µm wide0.5µm thickpitch 250µm

Nature Nanotech. 1 (2006) 214.

© CRANN www.crann.tcd.ie

DN

A hybridization

Driving personalized diagnostics through nanomechanics

J. Zhang et al., Nature Nanotech. 1 (2006) 214.

Exploiting regulatory processes of the interferon-alpha inducible gene 1-8U for cancer treatment using cantilever arrays at picomolar sensitivity levels.

Gene Idle Gene Upregulated

Nanomechanics in Biology

Static mode

© CRANN www.crann.tcd.ie

DN

A hybridization

Driving personalized diagnostics through nanomechanics

J. Zhang et al., Nature Nanotech. 1 (2006) 214.

Exploiting regulatory processes of the interferon-alpha inducible gene 1-8U for cancer treatment using cantilever arrays at picomolar sensitivity levels.

Gene Idle Gene Upregulated

Nanomechanics in Biology

Static mode

© CRANN www.crann.tcd.ie

DN

A hybridization

Driving personalized diagnostics through nanomechanics

J. Zhang et al., Nature Nanotech. 1 (2006) 214.

Exploiting regulatory processes of the interferon-alpha inducible gene 1-8U for cancer treatment using cantilever arrays at picomolar sensitivity levels.

Gene Idle Gene Upregulated

Nanomechanics in Biology

No LabelNo PCR AmplificationSensitivity ~ 10 fMSelectivity (Species, SNP)ncRNA direct measurementsfrom lysed cells or serum

Static mode

© CRANN www.crann.tcd.ie

Quantitative real-time measurements of virus binding to native bio-membranes

System under investigation: Bacterialvirus binding to membrane embeddedreceptor

Detection of mass uptake (nano grams)

Bio-functional microcantilevers detect viruses binding to membrane proteins in liquidCurrent sensitivity 30fM -> 1 virus / 4 cells

200 µm

sens

ref

time

T. B

raun

/ C

RA

NN

Nat

ure

Nan

otec

h. 4

, 179

-185

Proteomics

© CRANN www.crann.tcd.ie

Quantitative real-time measurements of virus binding to native bio-membranes

System under investigation: Bacterialvirus binding to membrane embeddedreceptor

Detection of mass uptake (nano grams)

Bio-functional microcantilevers detect viruses binding to membrane proteins in liquidCurrent sensitivity 30fM -> 1 virus / 4 cells

200 µm

sens

ref

time

T. B

raun

/ C

RA

NN

Nat

ure

Nan

otec

h. 4

, 179

-185

© CRANN www.crann.tcd.ie

Fast micro-organism growth detection

Target microorganisms: fungi, eukaryotes, bacteria, …

Microorganism play an important role in causing diseases, food spoiling, contamination of agriculture production and bio-destruction of substances in industry. Therefore a fast detection method of living micro-organism is of great importance. Hospital analysis is providing identification of microbe after 4 days, based on nutritional media.

N. Maloney, G.Lukacs / CRANN

Micro-/Cellbio

© CRANN www.crann.tcd.ie

Fast micro-organism growth detection

Target microorganisms: fungi, eukaryotes, bacteria, …

Microorganism play an important role in causing diseases, food spoiling, contamination of agriculture production and bio-destruction of substances in industry. Therefore a fast detection method of living micro-organism is of great importance. Hospital analysis is providing identification of microbe after 4 days, based on nutritional media.

N. Maloney, G.Lukacs / CRANN

© CRANN www.crann.tcd.ie

Hospital challenge• Identity after 4 days, based on incubation

condition, Highly skilled personnel

Fast Microbial Growth DetectionIndustrial ChallengeDisadvantages of current methods used inQuality and Safety procedures in industry:

• Time consuming: Analysis time > few days• Live/dead cell discrimination• Requires highly skilled personnel

25 mL agar mediumThickness 5 mmPlate diameter 10cm

10-8 mLnutritive layer

Cantilever dimensions: Length: 500 µm;Width: 100 µm; Thickness: 2-7 µm; Pitch: 250 µm;

A. niger C. albicans

E. coli P.aeruginosa

S.aureus

Microorganisms grown oncantilever sensors @ CRANN

G. Lukacs, N. Maloney

ESBL in hospitals

Extended spectra beta lactamase (ESBL) resistance transmitted to people via food uptake, increase of resistance in western countries more than 10x during the last five years

Growth Detection Time

ProkaryotesEukaryotes

© CRANN www.crann.tcd.ie

Hospital challenge• Identity after 4 days, based on incubation

condition, Highly skilled personnel

Fast Microbial Growth DetectionIndustrial ChallengeDisadvantages of current methods used inQuality and Safety procedures in industry:

• Time consuming: Analysis time > few days• Live/dead cell discrimination• Requires highly skilled personnel

25 mL agar mediumThickness 5 mmPlate diameter 10cm

10-8 mLnutritive layer

Cantilever dimensions: Length: 500 µm;Width: 100 µm; Thickness: 2-7 µm; Pitch: 250 µm;

A. niger C. albicans

E. coli P.aeruginosa

S.aureus

Microorganisms grown oncantilever sensors @ CRANN

G. Lukacs, N. Maloney

ESBL in hospitals

Extended spectra beta lactamase (ESBL) resistance transmitted to people via food uptake, increase of resistance in western countries more than 10x during the last five years

Growth Detection Time

ProkaryotesEukaryotes

© CRANN www.crann.tcd.ie

Hospital challenge• Identity after 4 days, based on incubation

condition, Highly skilled personnel

Fast Microbial Growth DetectionIndustrial ChallengeDisadvantages of current methods used inQuality and Safety procedures in industry:

• Time consuming: Analysis time > few days• Live/dead cell discrimination• Requires highly skilled personnel

25 mL agar mediumThickness 5 mmPlate diameter 10cm

10-8 mLnutritive layer

Cantilever dimensions: Length: 500 µm;Width: 100 µm; Thickness: 2-7 µm; Pitch: 250 µm;

A. niger C. albicans

E. coli P.aeruginosa

S.aureus

Microorganisms grown oncantilever sensors @ CRANN

G. Lukacs, N. Maloney

ESBL in hospitals

Extended spectra beta lactamase (ESBL) resistance transmitted to people via food uptake, increase of resistance in western countries more than 10x during the last five years

Growth Detection Time

Conventional MethodsCL Array ~ 1.5 hrs

~ 8 hrs

~4 hrsCL ArrayConventional Methodsµ-Fungi ~4-6days

ProkaryotesEukaryotes

© CRANN www.crann.tcd.ie

Nanomechanical Sensors for Quantitative Infection Detection

• By scaling mechanical sensors to the micron or nano regime enormous sensitivities can be achieved

• Direct nano-mechanical quantitative label-free detection of viable species within < 1 hr

• Differential read-out mandatory (in situ control measurement)• Stress measurements 1000 x more sensitive than SPR• Form factors: Cantilever, Double Clamped Beam, Hollow Beam, Squares• Automation possible (ink-jet spotting) allows spotting of successive layers• Interdisciplinary approach required• Among current projects: Malaria vaccination efficacy assay (STI), RNAi

diagnostic (Roche), Q&S (Novartis), Integration (CalTech).

Species which cause infections: Virus and microorganismsGenomic ncRNA biomarker detection

© CRANN www.crann.tcd.ie

Nanomechanical Sensors for Quantitative Infection Detection

• By scaling mechanical sensors to the micron or nano regime enormous sensitivities can be achieved

• Direct nano-mechanical quantitative label-free detection of viable species within < 1 hr

• Differential read-out mandatory (in situ control measurement)• Stress measurements 1000 x more sensitive than SPR• Form factors: Cantilever, Double Clamped Beam, Hollow Beam, Squares• Automation possible (ink-jet spotting) allows spotting of successive layers• Interdisciplinary approach required• Among current projects: Malaria vaccination efficacy assay (STI), RNAi

diagnostic (Roche), Q&S (Novartis), Integration (CalTech).

Species which cause infections: Virus and microorganismsGenomic ncRNA biomarker detection

Nano-BioMEMSBasic Science

Applied Science

© CRANN www.crann.tcd.ie

Nanomechanical Sensors for Quantitative Infection Detection

• By scaling mechanical sensors to the micron or nano regime enormous sensitivities can be achieved

• Direct nano-mechanical quantitative label-free detection of viable species within < 1 hr

• Differential read-out mandatory (in situ control measurement)• Stress measurements 1000 x more sensitive than SPR• Form factors: Cantilever, Double Clamped Beam, Hollow Beam, Squares• Automation possible (ink-jet spotting) allows spotting of successive layers• Interdisciplinary approach required• Among current projects: Malaria vaccination efficacy assay (STI), RNAi

diagnostic (Roche), Q&S (Novartis), Integration (CalTech).

Species which cause infections: Virus and microorganismsGenomic ncRNA biomarker detection

Nano-BioMEMSBasic Science

Applied Science

Label-free mixed assays in liquid environment possible with nanomechanical cantilever array

sensors

© CRANN www.crann.tcd.ie

The Nanomechanics Project Team@ CRANN, Trinity College Dublin

D. BrüggemannS. CullenV. JadhavJ. JensenG. LukacsN. MaloneyR. MischraM. WaltherF. Wruck

Former members:V. Aranyos, Y. Arntz, N. Backmann, S.-L. Ball,V. Barwich, M. Baller, F. Battiston, M. Bell,P. Bertoncini, A. Bietsch, T. Braun, A. Breedekamp, R. Daly, U. Dammer, M. D’Andrea, M. Dreier,M. Dorrestijn, M.Farina, J. Fritz, S. Garry, H. Gaussier,W. Grange, K. Gfeller, M. K. Ghatkesar, P. Haas,F. Huber, S. Husale, R. Joshi, M. Karle, M. McKane,R. McKendry, N. McLaughlin, P. Noy, N. Nugaeva,L. O’Connel J.P. Ramseyer, E. Rebourt, K. Renggli, P. Shahgaldian I. Schumakovitch, D. SkokoT. Strunz, M. Vögtli, G. Yoshikawa, J. Zhang

In collaboration with:Roche Center of Molecular GeneticsU. Certa, SwitzerlandCalTech USAM. RoukesFZ Jülich GermanyG. BüldtSwiss Tropical InstituteC. Daubenberger, SwitzerlandUniversity of BonnW. Voos, GermanyTrinity College DublinY. G’unko, G. Duesberg, IrelandNovartisM. Schuleit, SwitzerlandSt. James HospitalT. Rogers, IrelandUniv. Paris IV, Inst. Jacques MonotW. Grange, France

M. Hegner