Dai biomateriali ai sistemi olfattivi artificiali per la caratterizzazione di

campioni biologici

Arnaldo D’Amico, Marco Santonico, Giorgio Pennazza

XCVIII Congresso Nazionale Napoli, 17-21 Settembre 2012

About us

Roma Downtown

Fiumicino airport Roma - Napoli

highway

Tor Vergata University

GRA highway

Campus Bio-Medico

Laurentina

ROME

Outline

Introduction

Sensors and nanosensors definitions

Chemical interactive materials

Artificial olfactory systems

Medical applications

About us

• Tor Vergata University • Campus Bio-Medico • Founded in 1980

• 6 faculties

– Human sciences; Engineering, Sciences, Economics, Law, and Medicine

• ≈ 20000 students

• The largest campus in Italy

Founded in 1993

2 faculties

Medicine and Biomedical Engineering

As university it is a unique case in Italy promoting a close collaboration between Physicians and Engineers

SENSORS AND NANOSENSORS: OVERVIEW

Quantities

physical

chemical

biological

nanosensors

sensors

Environmental

Bio- suffix: Devices having the

sensing part made by biological

material

Scale [m]

1

10-9

10-6

10-3

Interactions

Physical sensors:

Devices able to sense

physical quantities.

Example: acceleration

Sensors and Sensors and nanosensorsnanosensors definitionsdefinitions

Physical nanosensors:

Nanodevices able to sense

physical quantities

ST three-axis accelerometer

(A Sanz-Velasco et al. 2006 Solid-State

Electron. 50 S865)

Nanoindentor:The force range is up

to 500 µN and 1 mN for the two main

designs, with a force resolution of to

0.3 µN.

Example: Force

Nanotube: force sensors

Chemical sensors: Devices

able to sense chemical

quantities.

Sensors and Sensors and nanosensorsnanosensors definitionsdefinitions

Chemical nanosensors:

Nanodevices able to sense

chemical quantities.

CMOS

MOSFET(Pd)

Nanosensors used

to measure cancer

biomarkers in whole

blood.

Nanowire Pd

Biological sensors

devices able to sense

biological quantities.

Biological nanosensors

Nanodevices able to sense

biological quantities.

Surface plasmon resonance detection of

ligand binding

Localized plasmon resonance

Plasmon resonance

Sensors and Sensors and nanosensorsnanosensors definitionsdefinitions

BBIOSENSORSIOSENSORS DEFINITIONDEFINITION Biosensors: devices having the sensing part made

by biologic materials;

Chemical biosensors Devices

having the sensing part made by

biologic materials and sensitive

to chemical quantities. Dioxin sensor

Biological biosensors

Devices having the

sensing part made by

biologic materials and

sensitive to

biomaterials. Penicillin sensitive Enzyme

modified FET (EnFET)

Physical biosensors Devices having

the sensing part made by biologic

materials and sensitive to physical

quantities.

Pressure sensitivity of luminescent porphyrins: pressure

sensitive paint

NNANOSENSORSANOSENSORS DDEFINITIONEFINITION Nanobiosensors: Nanodevices having the sensing part

made by biologic materials

Chemical nanobiosensors

Nanodevices having the sensing

part made by biologic materials

and sensitive to chemicals. Example:Heavy metal detection by peptide

modified SWNT based FET Sensitivity to Ni2+

Biological nanobiosensors:

nanodevices having the

sensing part made by biologic

materials and sensitive to

biomaterials.

Physical nanobiosensors:

Nanodevices having the

sensing part made by

biologic materials and

sensitive to physical

quantities.

Diazotation of sulfanilic acid with NO2 at slopes on (010)

SNOM image

SSENSORSENSORS ANDAND NANOSENSORSNANOSENSORS: : OVERVIEWOVERVIEW

SSENSORSENSORS ANDAND NANOSENSORSNANOSENSORS: : OVERVIEWOVERVIEW

Many devices are today available to induce a property change (r, s, ..) from

a change of concentration of a given volatile compound

CCHEMICALHEMICAL INTERACTIVEINTERACTIVE MATERIALSMATERIALS

Numerous may be the effects deriving from the interactions between

the CIM and the Enviroment:

heat generation followed by a temperature increase;

changes of one of the following parameters: electronic charge,

mass, conductivity, refractive index, work function, photon

emission.

Varieties of CIMs are available for chemical and biosensing such as:

metal oxide semiconductors (SnOx,TiOx,Ta2O5,IrOx,WO2..), Metals

(Pt,Pd,Ni,Ag,Cr,Sb,K,);

ionic conductors (ZrO2, LaFx, CaFx, CeO2, Nasicon);

polymers (polipirrole, poliphenilacetilene, cellulose, poliuretane,

policarbonate, porphyrines, phtalocianines, polisiloxenes,..);

enzimatic systems (glucose-oxidase, lattosio-oxidase, urease, anti-

IgG, anilisteria,..)

Sensing:

CIM Transducing:

QMB Metallo

porphyrins

Film deposition

Molecular

film molecule

s

qq

mA

ff D×-=D

rm

2

0

02

TTOROR VVERGATAERGATA ELECTRONICELECTRONIC NOSENOSE

Olfactive neuron::

many non specific many non specific

receptors receptors

of the same kindof the same kind

Artificial olfaction sensor::

many non specific many non specific

Receptors Receptors

of the same kindof the same kind

L. Buck and R. Axel; Cell 1991

N N

N N

N N

N N

N N

N N

N N

N N

OOLFACTIVELFACTIVE MAPSMAPS COMPARISONCOMPARISON

WWHYHY GASGAS SENSORSENSOR ARRAYARRAY??

It emerges that there is a number of molecular families whose alteration in concentration may be related to the presence of specific diseases.

The presence of the disease then produces a pattern of VOCs that are distinct (in concentration) from that found in healthy subjects.

This is a typical situation where a chemical sensor array can be applied.

VOCVOCSS ANALYSISANALYSIS ININ MEDICINEMEDICINE

Metabolic profile

Basic cellular functions including maintenance of cell membrane integrity, energy metabolism and especially oxidative stress are all known to be linked with VOC formation.

(Horvath et al., Eur Respir J 2009; 34: 261–

275)

VOCVOCSS ANALYSISANALYSIS ININ MEDICINEMEDICINE

D'Amico, A., et al. Detection and identification of cancers by the electronic nose. 2012. Expert Opinion on

Medical Diagnostics 6 (3) , pp. 175-185

TTOROR VVERGATAERGATA--CCAMPUSAMPUS BBIOIO--MEDICOMEDICO DIDI RROMAOMA

MEDICALMEDICAL APPLICATIONSAPPLICATIONS

DISEASE ANALYTE MEDICAL PARTNER

(ALL BASED IN ROME)

Schizophrenia sweat Italian Hospital Group

Lung cancer breath C. Forlanini Hospital

Asthma breath Catholic University of

Sacred Hearth

COPD breath Campus Bio-Medico di Roma

Bladder cancer urine Villa Pia Private Hospital

Prostate cancer urine “Tor Vergata” Hospital

Breast cancer skin San Eugenio Hospital

Melanoma skin Italian Dermopathic

Institute

Tumor cells :

in vitro and xenografted on animal

models

headspace Sant’Andrea Hospital

LLUNGUNG CANCERCANCER

Lung cancer studies with gas sensor arrays

2012

2010

LUNG CANCER Five research groups

Tor Vergata University,

Rome, Italy

Campus Bio-Medico di Roma

Rome, Italy

The Cleveland Clinic,

Cleveland, Ohio, United States

Zhejiang University, Hangzhou, China

Israel Institute of Technology, Haifa, Israel

Academic Medical Center, Amsterdam, Netherlands

LUNG CANCER, 2012

LUNG CANCER, 2012

METHOD

For

VOCs extraction

Via

endoscopy

Lung Cancer, 2012

Population Classification

NEGATIVE vs CANCER

ADK vs SCC

PPEOPLEEOPLE WITHWITH AASTHMASTHMA

Asthma is a common life-long chronic disease characterised by inflammation and narrowing of the airways. The narrowing does not occur all the time in mild asthma, but it happens more often as asthma gets more severe. It may also vary over short periods of time by itself or as a result of treatment

Symptoms can be controlled Symptoms of asthma include: Wheezing shortness of breath chest tightness and cough. Symptoms improve with appropriate treatment, so much so that treatment fails to control symptoms in only 5% of patients.

•Breath sampling for dead space (blu)

•Alaveolar space (red)

Alveolar volume segregation:Alveolar volume segregation:

ENOSEENOSE--GCGC--MS sampling protocolMS sampling protocol

Montuschi et al., Chest 2010

ENOSEENOSE--GC/MS: SGC/MS: SAMPLINGAMPLING PROTOCOLPROTOCOL

Total volume

Alveolar

breath

Montuschi et al., Chest 2010

COMPARISON OF DIFFERENT TECNIQUES

USED FOR ASTHMA DIAGNOSIS

Montuschi et al., Chest 2010

AASTHMASTHMA: GC: GC--MS MS RESULTSRESULTS

Principal component analysis (PCA) of mass spectrometry fingerprinting of patients with asthma and healthy subjects

Montuschi et al., Chest 2010

COPD DIAGNOSIS

Raffaele, Antonelli Incalzi, et al, Reproducibility and respiratory

function correlates of exhaled breath fingerprint in chronic

obstructive pulmonary disease , PLoS ONE (2012) in press

o Capuano, R., Santonico, M., Martinelli, E., Pennazza, G.,

Paolesse, R., Bergamini, A., et al. (2010). COPD diagnosis

by a gas sensor array. Paper presented at the Procedia

Engineering, , 5 484-487

COPD in elderly

REPRODUCIBILITY CONTROL INDIVIDUAL

COPD in elderly

REPRODUCIBILITY GOLD 4

COPD in elderly

TEST

CONFUSION MATRIX

predicted

0 1-2-3 4

real 0 5 0 0

1-2-3 0 12 3

4 0 0 5

Melanoma

Scientific background

Melanoma

Experiment

flow-chart

Melanoma

Measure phase

Cleaning phase

General overview

Sampling protocol

Melanoma

Measure strategy

Melanoma

1 2 3 4 5 6 7

0

20

40

60

80

100

120

140

Hz

Sensors

1 2 3 4 5 6 7

0

20

40

60

80

100

120

140

Hz

Sensors

diffe

rential df

nevi boxplot melanoma boxplot

BOXPLOTS OF ENOSE PATTERNS

diffe

rential df

Ref. Bernabei, M., Pennazza, G., Santonico, M., Corsi, C., Roscioni, C., Paolesse, R., et al. (2008). A preliminary study on the possibility to diagnose urinary tract cancers by an electronic nose. Sensors and Actuators, B: Chemical, 131(1), 1-4.

Analyte : urine

BLADDER CANCER

TNM Classification

Jewett-Strong Marshall

Definition

Tis 0 Limited to mucosa, flat insitu

Ta 0 Limited to mucosa, papillary

T1 A Lamina propria invaded

T2a B1 < halfway through muscularis

T2b B2 > halfway through muscularis

T3 C Perivesical fat

T4a C Prostate, uterus or vagina

T4b C Pelvic wall or abdominal wall

N1-N3 D1 Pelvic lymph node(s) involved

M1 D2 Distant metastases

Classification proposed by Jewett on 1946 and

revised by Marshall in 1956 (American Urologic

System).

Extr. From: http://training.seer.cancer.gov/

35-47%

50 %

(5%)

95%

recidivism mortality

development of disease and mortality

STATISTICAL

0: control

1: disease

6: post surgical

-0.15 -0.1 -0.05 0 0.05 0.1 0.15 0.2-0.2

-0.15

-0.1

-0.05

0

0.05

0.1

0.15

1 1

1

1

1 1

1 1

1

1

1

1 1 1

1 1 1

1 1 1

1 1

1 1 1 1 1

1 1

1 6 6

1

1

1 1

1

1 1 1 1

1

1

1

1

1 1 1 1 1 1 1 1

1 1 6 6 6

6 6 6

1

1

1 1

1

6 6

1 1

1

1 1

1 1 6 6

1 1 1 1 1 1

1

1

1 1 1 1 1

1

1 1

1 1 1 1 1

1

1

1

1

1

1 1

1 1 1 1 1 1

6 6

6 0 0 0

0 0 0 0

0

0 0

0

0

0

0 0 0

0 0

1

PC 1 (50.19%)

PC

2 (

27.5

4%

)

Scores Plot

desease

Healthy

Post surgery

patients

Scores plot of the first two components of PCA model

ELECTRONIC NOSE RESULTS

Analyte : urine

A NOVEL APPROACH FOR

PROSTATE CANCER

DIAGNOSIS USING A GAS

SENSOR ARRAY

Digital rectal

examination is the

first diagnostic

approach

transrectal

ultrasound Biopsy Prostate Specific

Antigene

Diagnostic Diagnostic iteriter

Non invasive

approach

Canine

olfaction

Invasive

approach

Artificial olfactory system

Pilot study:primliminary resultsPilot study:primliminary results

ENOSE

reference

sample

200 sec 600 sec

Scores plot of the first two

latent variables obtained

by PLS-DA model.

The result has been

obtained considering the

first part of urine.

In this case two control

subjects have been

misclassified as ill

subjects.

D'Amico, A., et al. Detection and identification of cancers by

the electronic nose. 2012. Expert Opinion on Medical

Diagnostics 6 (3) , pp. 175-185