Miniaturized Capacitive Devices for the Detection of Circulating ... · Micro&Nano Carbon Lab Tomo...

DDF Summit, 11-13 March 2019, Berlin

Miniaturized Capacitive Devices for the

Detection of Circulating Biomarkers, Enabling

Real-Time Kinetic Measurements, Integrable

with Microfluidic Systems

Federica Mantovani

Loredana Casalis

Berkeley, 22 February 2018

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Elettra Sincrotrone Trieste

Elettra Sincrotrone Trieste is a multidisciplinary international

centre of research, specialized in synchrotron and free-

electron laser light for materials and life science.

No profit shareholder company recognized of national interest.

Basic and Applied Research

Technical and scientific training

Transfer of technology and

know-how

• 400 employees

• 34 beamlines

• 12 support lab

• 5000 hours /

year

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1000 users

from more

than 50

countries

Syncrothron Elettra: 28 beamlines

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BoosterAnello di

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di misura

Elettra is a ʺpowerful miscroscopeʺ, using a particular type of radiation to carry out measurements and analysis of materials, the syncrothron light

28 beamlines at

least 28 measurement

techniques!

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❖ Wavelength is tunable according to the material and characteristic to be

explored

❖ Very bright light

❖ Coherent and collimated light ray

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Elettra

Experimental techniques

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Life Sciences @Elettra

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BeamlinesLaboratories

Micro&Nano Carbon Lab

Tomo LabX-ray computed micro tomography system based on a micro focus source.

Nanoinnovation Labsurface confined bio-molecules; self-assembled monolayers; atomic force microscopy; nano-biomedicine

Structural Biology Labbiophysical, biochemical characterization of proteinscrystallization, 3D structure determination,high-throughput protein production

preparation and study of carbon nanotubes and carbon based materials

XRD1/XRD2/MCXX-ray diffraction on single crystals; powder diffraction

SAXSSmall-angle X-ray scattering; grazing incidence (GISAXS); Wide-angle scattering (WAXS).

SISSIInfrared spectroscopy; FTIR; imaging

SYRMEPMedical diagnostic radiology; phase-contrast imaging; micro tomography

TwinMicX-ray microscopy; combined scanning and full-field imaging modes


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❖ Liquid biopsy: the quest for miniaturized, point-of-care devices

❖ Protein Nanoarrays for liquid biopsy: Her2 detection

❖ Real-time capacitive devices for RNA/DNA detection

❖ Real-time capacitive devices for circulating protein detection

Miniaturized Capacitive Devices for the Detection of Circulating

Biomarkers, Enabling Real-Time Kinetic Measurements, Integrable

with Microfluidic Systems


Layout

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Miniaturized sensors for liquid biopsy

Liquid Biopsy

Both healthy and tumor (diseased) cells transmit information on their

state to the bloodstream, either directly (proteins) or as cell signaling

intermediate (exosomes, metabolites).

Such information are possible biomarkers for early non-invasive

population screening, cancer diagnosis and prognosis and for

monitoring the response to therapeutic treatment.

M. Westpahl, Nature Review Neurology, 2015

C. Dincer et al., CellPress 2017

Emerging application in resource-

limited settings: early and accurate

diagnosis


Our nanotechnology based devices

❖ Protein nanoarrays

▪ ultimately enabling single cell proteomic analysis

❖ Capacitive immunosensors

▪ enabling real-time, kinetic measurements.

▪ Integrable with microfluidic systems

We have developed two classes of miniaturized devices:


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Human Epidermal Growth Factor Receptor 2 (Her2) is a member of the epithelial growth factor receptor

(EGFR/ErbB) family; overexpression of Her2 has be found in breast, ovarian and gastric cancers with high

frequencies

P P

Dimerization process leads to the proteolytic release of

Her2 Extra-Cellular Domain (ECD)

❖ ECD acts as ligand for other transmembrane protein

❖ Proteolytic release of ECD-Her2 in the bloodstream is

an important indicator of tumoral potential

❖ the amino-terminally truncated membrane-associated

fragment increases significantly its tyrosine kinase

activityLIQUID BIOPSY

The dimerization leads to the activation of

different downstream signaling cascades

involved in cell proliferation and cancer

progression

Circulating cancer biomarkers.

Case study: Her2 positive tumors

DDF Summit, 11-13 March 2019, Berlin13

F. Bano, L. Fruk, C. Niemayer, L. Casalis, G. Scoles, et al., Nanoletters 2009M. Ganau, A. Bosco et al., Nanomedicine and Nanotechnology 2015E. Ambrosetti, E. Tagliabue, A. DeMarco, P. Parisse and L. Casalis, ACS Omega, 2017

ECD-Her2 early detection in serum using nanobodies (VHH)

Limit of

sensitivity150 pM Lower then the cutoff value of 15 ng/ml

(200 pM) used in clinic for Her2 positive

breast cancer assessment

Multiplexed detection

Nanodiagnostics platforms: nanoarraysDetection of ECD-Her2 with nanobodies

of different

biomarkers


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Microstructured impendance biosensorsReal-time devices for RNA/protein analysis

Starting from previously gained know how and

capabilities –nannoarrays as nanodiagnostics platform

(Her 2 detection and Therapeutic Drug Monitorning),

DNA-coated gold nanoparticles as colorimetric sensors

(helicase inhibitor screening)- a new sensor has been

developed:

❖ A easy-to-use device with electric read-out (Lab-

on-a-Chip technology-wise)

❖ Its use and sensitivity in diagnostics has been

explored and demonstrated

❖ Its use in Therapeutic Drug Monitoring does have a

high potential

❖ Its potential market as a dignostic kit is promising


Microstructured impendance biosensorsReal-time devices for RNA/protein analysis

In the hypothesis that Cstray

does not have any effect on

the current:

We fabricated via soft lithography gold microelectrodes

(working and counter electrodes, WE and CE) and

functionalized them with a Self-Assembled Monolayer of

ssDNA.

We characterized the interface in buffer solution, applying a

small (below 100 mV) AC potential between WE and RE, and

sampling the response of the system in the low frequency

regime (below 1 KHz).

By using a liquid cell with a standard electrophysiology

reference electrode RE (1 mm diameter), we then derived the

capacitance of the system using the equivalent circuit scheme:


Microstructured capacitance biosensorsReal-time devices for RNA/protein analysis

For low density ssDNA SAMs (the ones giving

higher hybridization efficiency) we can think of

different contribution to the system capacitance

given by Cmol and Cions at the functionalized WE

Following DNA hybridization, Cmol changes

because of:

❖ the increased charge density within the DNA

monolayer

❖ the increase height of the film due to the

different persistence length of ssDNA (1 nm)

and dsDNA (50 nm)


Real-time analysis of DNA hybridization

L. Ianeselli, G. Grenci, C. Callegari, M. Tormen and L. Casalis et al., Biosensors and Bioelectronics 2014

P. Capaldo, S. Alfarano, A. Bosco, P. Parisse, L. Casalis, ACS Sensors 2016

10 pM DNA conc

DNA binding affinity curve

Kinetic of DNA hybridization via differential capacitance

measurement

Limit of Detection: 1pM


Real-time analysis of DNA hybridizationReal-time analysis of DNA hybridizationBase mismatch detection

P. Capaldo and M.D. Nkoua Ngavouka, E. Ambrosetti, L. Casalis , P. Parisse. Beilstein J. Nanotechnol., 2016

focus on selectivity


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Real-time ECD-Her2 analysis in standardized human serum(Her2+ Breast cancer)


Real-time ECD-Her2 analysis in standardized human serum(Her2+ Breast cancer)

KD= (2.15±0.97) nM

KD(SPR)≈ 1.7 nM


Future developments

Future plan: multiplexed array of coplanar electrodes

with microfluidics and hydrophobic valves


Acknowledgments & contacts

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❖ Special thank you to Prof. Loredana Casalis e her NanoinnovationLab group

❖ For further questions:

[email protected]

[email protected]

mailto:[email protected]

mailto:[email protected]

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