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Sensitive Molecular Diagnostics with Electrochemical Biosensors and Ensembles of Nanoelectrodes

Sensitive Molecular Diagnostics with Electrochemical Biosensors and Ensembles of Nanoelectrodes. Silvestrini M , Ugo P, De Leo L, Not T, Žiberna L, Passamonti S, Martelanc M, Franko M. Sensitive Molecular Diagnostics with Electrochemical Biosensors and Ensembles of Nanoelectrodes.

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Sensitive Molecular Diagnostics with Electrochemical Biosensors and Ensembles of Nanoelectrodes

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  1. Sensitive Molecular Diagnostics with Electrochemical Biosensors and Ensembles of Nanoelectrodes Silvestrini M, Ugo P, De Leo L, Not T, Žiberna L, Passamonti S, Martelanc M, Franko M

  2. Sensitive Molecular Diagnostics with Electrochemical Biosensors and Ensembles of Nanoelectrodes What is an electrochemical Biosensor? Analyte in a complex matrix FIRST GENERATION Recognition Transduction Biorecognition layer Transducer (electrode) SECOND GENERATION Electrical signal Signal processing THIRD GENERATION

  3. Laboratory of Sensors for Electroanalysis Bio-electrochemical sensors • Preparation 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).

  4. Sensitive Molecular Diagnostics with Electrochemical Biosensors and Ensembles of Nanoelectrodes Fabrication and analytical application of ensembles and arrays of nanoelectrodes, nanostructured electroactive membranes and polymer-based electrodes. Nanoelectrode ensembles (NEEs) Nanoelectrode arrays (NEAs)* * In collaboration with TASC-Trieste. P. Ugo et al. Chemphyschem. 3 (2002), 917. L. M. Moretto et al. Nanotechnology22 (2011), 185305. P. Ugo, L. M. Moretto, in Handbook of Electrochemistry (1st edn.), edited by C.G. Zoski, Elsevier (2007) (Chapter 16.2).

  5. Gold 10-250 nm Track-etched membrane 1-6 m Sensitive Molecular Diagnostics with Electrochemical Biosensors and Ensembles of Nanoelectrodes Ensembles of Nanoelectrodes (NEEs) V.P. Menon, C.R. Martin, Anal. Chem. 67 (1995), 1920.

  6. Sensitive Molecular Diagnostics with Electrochemical Biosensors and Ensembles of Nanoelectrodes 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. High sensitivity in small volume samples. Detection of µM-nM concentrations, in 10-100 µL volumes of turbid raw samples (= pico-moles of analyte). Electrochemical transduction of signals is: -simple - unaffected by light, turbidity etc. - does not require microscopy - instrumentation is cheap & transportable.

  7. - - - - - - - - e + + + + + + Biosens. Bioelectron. 23 (2008), 1900. Electroanalysis19 (2007), 227. Anal. Chem. 68 (1996), 4160. Sensitive Molecular Diagnostics with Electrochemical Biosensors and Ensembles of Nanoelectrodes Functionalization of electrochemical nanoelectrode ensembles (NEEs) Coatingonly the nanoelectrodesafteretching Coatingall the ensemble Functionalization of the membrane

  8. HRP H2O2 Anti-mouse-HRP sec-Ab HER2 HER2 receptor 0. 1 0. 2 0. 3 0. 4 HER2 [ng/L] Sensitive Molecular Diagnostics with Electrochemical Biosensors and Ensembles of Nanoelectrodes 2D-NEEs functionalizedwithTrastuzumab (T-NEEs) HerceptinTM (trastzumab) is a monoclonal Ab active against breast cancer characterized by overexpression of Her2 receptor. Increaseofpeakcurrents at T-NEE in celllysatescontaining HER2 receptor H2O MB LB PrimaryAb HerceptinTM (trastuzumab) e- Increaseofsignal at low concentration. Saturation at high concentration. Lowerconcentrationdetected 0.4 ng. DL Western Blotapprox. 1 ng. Cell lysates containing the target protein were diluted 1:5, 1:10 and 1:50. S. Pozzi Mucelli, M. Zamuner, M. Tormen, G. Stanta, P. Ugo, Biosens. Bioelectron. 23, 1900 (2008)

  9. 2D-NEEs for DNA detection FA+ GOxox Glucose 2e- NEE GOxred FA2+ Gluconolactone With glucose (50 mM) Without glucose M. Silvestrini, L. Fruk, P. Ugo, Biosens. Bioelectron. 40 (2013), 265.

  10. Sensitive Molecular Diagnostics with Electrochemical Biosensors and Ensembles of Nanoelectrodes 3D-NEEs byimmobilizationofAuNPs NEE treated with NPs SAMs of cysteamine Non-thiolated NEE treatedwithAuNPs Thiolated NEE treatedwithAuNPs Bare NEE NEE treated with NPs Bare NEE M. Silvestrini, P. Ugo, Anal. Bioanal. Chem.405 (2013), 995.

  11. 3D-NEEs for DNA detection FA+ GOxox Glucose 2e- NEE GOxred FA2+ Gluconolactone With glucose (50 mM) Without glucose M. Silvestrini, P. Ugo, Anal. Bioanal. Chem.405 (2013), 995.

  12. Sensitive Molecular Diagnostics with Electrochemical Biosensors and Ensembles of Nanoelectrodes Trans2Care researchactivity Collaboration with Institute of Child Health IRCCS “BurloGarofolo”, 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.)

  13. Enzyme tTG Tissue transglutaminase (tTG) Sensitive Molecular Diagnostics with Electrochemical Biosensors and Ensembles of Nanoelectrodes Trans2Care researchactivity • Electrochemical detection of the autoantibody anti-transglutaminase (anti-tTG): • First approach (LABEL DETECTION) e- Substrate Product Detection by Cyclic Voltammetry …still ongoing! Med SecondaryAb Med Anti-transglutaminase (anti-tTG) NEE

  14. Sensitive Molecular Diagnostics with Electrochemical Biosensors and Ensembles of Nanoelectrodes Trans2Care researchactivity • Electrochemical detection of the autoantibody anti-transglutaminase (anti-tTG): • Second approach (LABEL-FREE DETECTION) Detection by Electrochemical Impedance Spectroscopy …still ongoing! Anti-transglutaminase (anti-tTG) Tissue transglutaminase (tTG) tTG tTG Thiol SAM Flat Au

  15. Sensitive Molecular Diagnostics with Electrochemical Biosensors and Ensembles of Nanoelectrodes Trans2Care researchactivity Collaboration with University of Trieste - Dept. of Life Sciences and University of Nova Gorica - Laboratory of Environmental Research 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

  16. Sensitive Molecular Diagnostics with Electrochemical Biosensors and Ensembles of Nanoelectrodes Toxicity of Bilirubin Hyperbilirubinemia 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.

  17. Sensitive Molecular Diagnostics with Electrochemical Biosensors and Ensembles of Nanoelectrodes Trans2Care researchactivity AII 1 mM BR in DMSO containing 0.1 M TBABF4 as supporting electrolyte Scan rate 20 mV/s O2 removed from solution with N2 AI CI AI = oxidation of BR to biliverdin (BV) AII = oxidation of BV to purpurin (Pu) CI = reduction of BR M. Silvestrini et al., in preparation.

  18. Sensitive Molecular Diagnostics with Electrochemical Biosensors and Ensembles of Nanoelectrodes Preliminary study of the reactivity with electrogenerated O2- 0.1 M TBABF4 in DMSO with O2 Scan rate: 50 mV/s [BR] (mM): __ 0 __ 0.8 __2 O2 + e- ⇄ O2-• O2-• + HBR ⇄ BR- + O2H• O2-• + O2H• → HO2- + O2 HO2- + HBR⇄ BR- + H2O2 M. Silvestrini et al., in preparation.

  19. Sensitive Molecular Diagnostics with Electrochemical Biosensors and Ensembles of Nanoelectrodes Electrochemical detection of BR in serum Serum or plasma total BR Glucuronide-conjugated bilirubin (CB) Unconjugated bilirubin (UCB) Free bilirubin Bilirubin covalently bound to albumin 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!

  20. Thankyouforyourattention! Collaborationsacknowledged: AB Analitica srl (Padova); Veneto Nanotech – Nanofab (Venezia-Marghera); KIT-Frukgroup (Karlsruhe, Germany); Trans2Care Project Partners (LP, PP3, PP9). Laboratory of Sensors for Electroanalysis http://lsegroup.wix.com/website-lse-group 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. 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.

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