sensitive molecular diagnostics with electrochemical biosensors and ensembles of nanoelectrodes

Sensitive Molecular Diagnostics with Sensitive Molecular Diagnostics with Electrochemical Biosensors and Electrochemical Biosensors and Ensembles of NanoelectrodesEnsembles of Nanoelectrodes

Silvestrini MSilvestrini M, Ugo P, De Leo L, Not T, Žiberna L, Passamonti S, , Ugo P, De Leo L, Not T, Žiberna L, Passamonti S, Martelanc M, Franko MMartelanc M, Franko M

http://www.nanotechitaly.it/

Sensitive Molecular Diagnostics with Electrochemical Biosensors and Ensembles of Nanoelectrodes

What is an electrochemical Biosensor?

Analyte in a complex matrix

Biorecognition layer

Transducer (electrode)

Electrical signal

Recognition

Transduction

Signal processing

e-

Medred

Medox

Substrate

Product

e-Substrate

Product

electrode

electrode

e- Enzy

me Substrate

Product

electrode

e-O2

O2

H2O2

A)

B)

C)

Enzy

me

Enzy

me

FIRST GENERATION

SECOND GENERATION

THIRD GENERATION

Bio-electrochemical sensorsBio-electrochemical sensorsPreparation and characterization of sensors based on electrodes and arrays of electrodes of size from few nm to mm.

Direct electroanalysis of the redox enzymes such as Cytochrome c;

Electrochemical sensor for glucose analyses which employs glucose dehydrogenase and a special redox mediator (a nitrofluorenone), which is very efficient in catalyzing NADH oxidation (collaboration with University of Bordeaux 1);

Electrochemical immunosensors applied for the detection of the HER2 receptor. The HER2 receptor is an important target protein for the identification of cancer that can be treated successfully with Herceptin (Trastuzumab) by the so called personalized therapies (collaboration with ICGB-Trieste, prof. G. Stanta);

Recent development of new procedures suitable for immobilizing oligonucleotides on nanoelectrode ensembles (collaboration with AB-Analitica srl, Padua; CIVEN, Porto Marghera-Venezia; Karlsrue Institute of Technology - Dr. Ljljiana Fruk’s group).

Laboratory of Sensors for Electroanalysis

Fabrication and analytical application of ensembles and arrays of nanoelectrodes, nanostructured electroactive membranes and polymer-based electrodes.

P. Ugo et al. Chemphyschem. 3 (2002), 917.L. M. Moretto et al. Nanotechnology 22 (2011), 185305.P. Ugo, L. M. Moretto, in Handbook of Electrochemistry (1st edn.), edited by C.G. Zoski, Elsevier (2007) (Chapter 16.2).

* In collaboration with TASC-Trieste.

Nanoelectrode ensembles (NEEs) Nanoelectrode arrays (NEAs)*



V.P. Menon, C.R. Martin, Anal. Chem. 67 (1995), 1920.

Ensembles of Nanoelectrodes (NEEs)

Gold Track-etched membrane

1-6 m

10-250 nm

Advantages of electrochemical nanobiosensors

Wide possibilities for immobilizing biorecognition layers: enzymes, antibodies, DNA.

Because of the nano-geometry:- highly improved S/N ratio (2-3 order of magnitudes)

- suitable to extreme miniaturization.

Electrochemical transduction of signals is:-simple

- unaffected by light, turbidity etc.- does not require microscopy

- instrumentation is cheap & transportable.

High sensitivity in small volume samples. Detection of µM-nM concentrations, in 10-100 µL volumes of turbid raw samples

(= pico-moles of analyte).


Coating all theensemble

+ + + + + +

-- -- - -- -

Anal. Chem. 68 (1996), 4160.

Functionalization of the membrane

Coating only the nanoelectrodes

after etching

e

Electroanalysis 19 (2007), 227.Biosens. Bioelectron. 23 (2008), 1900.

Functionalization of electrochemical nanoelectrode ensembles (NEEs)


2D-NEEs functionalized with Trastuzumab (T-NEEs)

HRP

Anti-mouse-HRP sec-Ab

Primary Ab

HER2 receptor HER2

e-

H2O2

MBLB

HerceptinTM (trastuzumab)

H2O

HerceptinTM (trastzumab) is a monoclonal Ab active against breast cancer characterized by

overexpression of Her2 receptor.

Increase of peak currents at T-NEE in cell lysates containing HER2 receptor

0. 40. 30. 20. 1HER2 [ng/L]

Cell lysates containing the target protein were diluted 1:5, 1:10 and 1:50.

Increase of signal at low concentration.Saturation at high concentration.

Lower concentration detected 0.4 ng.DL Western Blot approx. 1 ng.


S. Pozzi Mucelli, M. Zamuner, M. Tormen, G. Stanta, P. Ugo, Biosens. Bioelectron. 23, 1900 (2008)

2D-NEEs for DNA detection

Gluconolactone

GOxox

GOxred

Glucose

FA2+

FA+ NEE

2e-

0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7

-0,2

0,0

0,2

0,4

0,6

0,8

1,0

I (A

)

E (V)

Without glucose

With glucose (50 mM)

C

C

O

OOH

OH

C

C

O

O

HN

HN

ssDNA

ssDNA

C

C

O

O

HN

HN

dsDNA

dsDNA

GOx

GOx

EDC/NHS ssDNA

Au

Au

Au

Au

Au

Au

PC PC PCssDNA-GOx

M. Silvestrini, L. Fruk, P. Ugo, Biosens. Bioelectron. 40 (2013), 265.

3D-NEEs by immobilization of AuNPs


SAMs of cysteamine

HSNH2

Non-thiolated NEE treated with AuNPs

-0,1 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8-0,08

-0,06

-0,04

-0,02

0,00

0,02

0,04

0,06

0,08

KNO3 1 mM, scan rate 50 mV/s

bare NEE NEE + AuNPs

I (uA

)

E (V)

NEE treated with NPs

Bare NEE

-0,1 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8-0,02

-0,01

0,00

0,01

0,02KNO3 1 mM, scan rate 50 mV/s

NEE27 non trattato NEE27 trattato con AuNPs senza cyst

I (uA

)

E (V)

Bare NEE

NEE treated with NPs

M. Silvestrini, P. Ugo, Anal. Bioanal. Chem. 405 (2013), 995.

Thiolated NEE treated with AuNPs

3D-NEEs for DNA detection

Gluconolactone

GOxox

GOxred

Glucose

FA2+

FA+ NEE

2e-

Without glucose

With glucose (50 mM)

HS- ssDNA

Au

PC

Au S NHS NH

S NHS NHAu

PC

Au S NHS NH

S NHS NH

S-ssDNAS-ssDNA

S-ssDNAS-ssDNA

ssDNA-GOx

Au

PC

Au S NHS NH

S NHS NH

S-dsDNA-GOxS-dsDNA-GOx

S-dsDNA-GOxS-dsDNA-GOx

-0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8-0.2

0.0

0.2

0.4

0.6

I (A

)

E (V)M. Silvestrini, P. Ugo, Anal. Bioanal. Chem. 405 (2013), 995.

Trans2Care research activityTrans2Care research activity


Collaboration with Institute of Child Health IRCCS “Burlo Garofolo”, Trieste

Fabrication of electrochemical biosensors for the detection of antibodies involved in autoimmune diseases,

directly in biological samples (i.e. blood, serum, intestinal biopsies).

Development of arrays of electrodes for the detection of different autoantibodies (e.g., anti-transglutaminase (anti-tTG), anti-gliadin, anti-

thyroid peroxidase (anti-TPO), etc.)



Substrate

Med

Product

e-

Med

Enzyme

Anti-transglutaminase (anti-tTG)

Tissue transglutaminase (tTG) tTG

Secondary Ab

Electrochemical detection of the autoantibody anti-transglutaminase (anti-tTG):

First approach (LABEL DETECTION)

Detection by Cyclic Voltammetry

…still ongoing!

NEE



Electrochemical detection of the autoantibody anti-transglutaminase (anti-tTG):

Second approach (LABEL-FREE DETECTION)

Detection by Electrochemical Impedance

Spectroscopy

…still ongoing!Flat Au

Thiol SAM

tTG tTG

Anti-transglutaminase (anti-tTG)

Tissue transglutaminase (tTG)

0 5000 10000 15000 20000 25000 300000

-2000

-4000

-6000

-8000

-10000

-12000

Impe

danc

e im

agina

ry p

art (

)Impedance real part ()


Development of an electrochemical sensor for bilirubin (BR) detection in serum

Electrochemical characterization of BR in organic solvent

Voltammetric detection of direct and total BR in serum samples


Collaboration with University of Trieste - Dept. of Life Sciences and University of Nova Gorica - Laboratory of Environmental Research

Toxicity of BilirubinHyperbilirubinemia is an excess of BR in the blood, occurring as a result of liver or biliary tract dysfunction or with excessive destruction of red blood cells. It is classified as conjugated or unconjugated, according to the type of bilirubin present. Jaundice is manifested when excess bilirubin is deposited in the skin and mucous membranes.

Hyperbilirubinemia in the newborn = excess of serum bilirubin in the newborn due either to overproduction of bilirubin, as in excessive destruction of erythrocytes, or to reduction in glucuronide conjugation in the liver. If a high level of bilirubinemia is left untreated, kernicterus may occur as a result of free unconjugated bilirubin entering the brain tissue and causing neurotoxic damage.




M. Silvestrini et al., in preparation.

1 mM BR in DMSO containing 0.1 M TBABF4 as supporting electrolyteScan rate 20 mV/sO2 removed from solution with N2

AIIAI

AI = oxidation of BR to biliverdin (BV)

AII = oxidation of BV to purpurin (Pu)

CI = reduction of BR

CI

0.1 M TBABF4 in DMSO with O2

Scan rate: 50 mV/s

[BR] (mM):__ 0__ 0.8__ 2

Preliminary study of the reactivity with electrogenerated O2-

O2 + e- O⇄ 2-•

O2-• + HBR BR⇄ - + O2H•

O2-• + O2H• → HO2

- + O2

HO2- + HBR BR⇄ - + H2O2


M. Silvestrini et al., in preparation.


Electrochemical detection of BR in serum

Serum or plasma total BR

Unconjugated bilirubin (UCB)

Glucuronide-conjugated bilirubin (CB)

Bilirubin covalently bound to albumin

Free bilirubin

Albumin-bound BR is electrochemically inactive.Serum total BR can be measured after precipitation of albumin.

Voltammetric detection of direct and total BR in serum samples with electrodes modified with ion conductive ionomers

…still ongoing!

Progetto Rete Transregionale per l’innovazione ed il Trasferimento Tecnologico per il Miglioramento della

Sanità finanziato nell’ambito del Programma per la Cooperazione Transfrontaliera Italia-Slovenia 2007-2013,

dal Fondo europeo di sviluppo regionale e dai fondi nazionali.

Projekt Transregionalno omrežje za inovacijo in prenos tehnološkega znanja za izboljšanje zdravstva

sofinancirana v okviru Programa čezmejnega sodelovanja Slovenija-Italija 2007-2013 iz sredstev Evropskega

sklada za regionalni razvoj in nacionalnih sredstev.

Partners: LP – Università degli Studi di Trieste, PP1 – Kemijski Inštitut Ljubljana, PP2 – Scuola Internazionale Superiore di Studi Avanzati, PP3 – Univerza v

Novi Gorici, PP4 – Università di Ferrara, PP5 – Treviso Tecnologia, PP6 – Splošna Bolnišnica Dr. Franca Derganca, PP7 – Università Ca’ Foscari di Venezia, PP8

– Università di Udine, PP9 – IRCCS Burlo Garofalo, PP10 – Zavod Republike Slovenije Za Transfuzijsko Medicino, PP11 – Ortopedska Bolnišnica Valdoltra,

PP12 – Univerza na Primorskem - Fakulteta za Vede o Zdravju.

Thank you for your attention!Thank you for your attention!

Collaborations acknowledgedCollaborations acknowledged: AB Analitica srl (Padova); Veneto Nanotech – Nanofab (Venezia-: AB Analitica srl (Padova); Veneto Nanotech – Nanofab (Venezia-Marghera); KIT-Fruk group (Karlsruhe, Germany); Trans2Care Project Partners (LP, PP3, PP9).Marghera); KIT-Fruk group (Karlsruhe, Germany); Trans2Care Project Partners (LP, PP3, PP9).

Laboratory of Sensors for Electroanalysishttp://lsegroup.wix.com/website-lse-group

http://lsegroup.wix.com/website-lse-group

sensitive molecular diagnostics with electrochemical biosensors and ensembles of nanoelectrodes

Documents

electrochemical biosensors

electrochemical immunosensors

high concentration

glucose analyses

glucose dehydrogenase

her2 receptorcell lysates

low concentration

lower concentration