BOOK of ABSTRACTS - UNIMORE · BOOK of ABSTRACTS GRUPPO DIVISIONALE SENSORI Università di Modena e Reggio Emilia 17 – 18 settembre 2009 ... essenziali per una corretta diagnosi

BOOK of

ABSTRACTS

GGRRUUPPPPOO DDIIVVIISSIIOONNAALLEE SSEENNSSOORRII

UUnniivveerrssiittàà ddii MMooddeennaa ee RReeggggiioo EEmmiilliiaa 1177 –– 1188 sseetttteemmbbrree 22000099

Università di Modena e Reggio Emilia

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ELECTRONIC NOSES FOR THE MEDICAL FIELD AND ELECTRONIC TONGUES FOR

WATER CHARACTERIZATION

Arnaldo D’Amico

Facoltà di Ingegneria, Dept. of Electronic Engineering University of Roma Tor Vergata

[email protected]

First this work will describe, in some detail, the operating principle and performances of the Tor Vergata electronic nose (EN) with some aspects related to the chemically sensitive materials which are represented by metalloporphyrins, and the configuration of the electronic tongue (ET), including electronic circuits as well. Then this presentation will report both an overview and recent applications in the medical field of the EN, and will illustrate some aspects of the electronic tongue in applications dealing with water analysis. In particular data obtained in the field of lung cancer disease detection will be commented with some details, as well as those related to melanoma studies without disregarding results related to the same samples analyzed by GC-MS technique. Performances of the ET will be discussed showing experimental results on varieties of waters. At the end the miniaturization process consequences of new sensors will be taken into consideration showing new aspects related to the information theory applied during adsorption-desorption elementary events.

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ABSTRACT

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COMUNICAZIONI

ORALI

NEW CALIBRATION MODELS FOR THE DETERMINATION OF MOL ECULAR OXYGEN WITH OPTICAL SENSORS BASED ON LUMINESCENCE Q UENCHING OF

TRANSITION METAL COMPLEXES EMBEDDED IN POLYMERIC MA TRICES

Denis Badocco1, Andrea Mondin, Alberto Fusar and Paolo Pastore

1 Dipartimento di Scienze Chimiche, Università di Padova, via Marzolo 1. [email protected]

Three oxygen sensitive polysulfone-based membranes employing ruthenium tris (4,7-diphenyl-l,l0-phenanthroline) octylsulfonate (Ru(dpp)OS),1 5,10,15,20-Tetraphenyl-21H,23H-porphyrin platinum(II) (PtTPP) and 5,10,15,20-tetrakis(pentafluorophenyl)-21H,23H- porphyrin palladium(II) (PdFTPP), were prepared and tested with different calibration approaches.2 The three luminescent labels were chosen for their different life-time giving the dimensionless SV constant, '

SVK , of

0.0176(0.0001), 0.146(0.001), and 1.768(0.014), for Ru(dpp)OS, PtTPP and PdFTPP, respectively.

The usual Stern-Volmer (SV) calibration approach (model (I): 2210 %1 OPPI

IRI ⋅+=−= ) was

compared with two alternative models based on the inflection point of the light emission profile (model (II): ( )221 %ln OPPflex ⋅+=γ v. Fig.1) and on a suitable integral of the light emission profile

(model (III): ( )92.0221 %1ln OPPA ⋅+⋅= ). Regression parameters P1 and P2 were determined and

their physical meanings were explained with digital simulation techniques enlightening the nature of the chosen approaches.3 Sensitivity, precision and working interval of the three membranes were studied. Model (I) fails for low oxygen percentage values, 2%O , (depending on luminophore

nature) whilst the other two works well in the whole 0-100 2%O range. Model (I) works up to 100%, 93% and 25% oxygen with Ru(dpp)OS, PtTPP and PdFTPP, respectively. Calibration sensitivities, S, are in the sequence AR SSS

I>> γ or AR SSS

I>>γ depending on the luminophore nature and

on the 2%O value. Model (II) is more sensitive than model (I) for %O2 <60%, <6% and <2%, for Ru(dpp)OS, PtTPP and PdFTPP membranes, respectively. Precision of the model (I) is almost constant as foreseen for a linear model. Averaged experimental precision values of 3.5, 0.7 and 0.4 % were found for Ru(dpp)OS, PtTPP and PdFTPP, respectively. A dimensionless kinetic constant, ( )09.025.2=γk , valid for all membrane typologies was determined. It allowed the

evaluation of the membrane thickness.

0 25 50 75 100

-2

0

2

4a

γ flex

%O2

PdFTPP

PtTPP

Ru(dpp)OS

0 10 200.0

0.5

1.0b

I/I0

time (s)

PdFTPP

1

15

Figure 1. a) Parametric regressions with prediction bands obtained with Model (II) for Ru(dpp)OS (○), PtTPP (□) and PdFTPP (∆). Regression parameters are reported in Table 2. b) Fitted profiles (bold line) overlapped to the experimental data of PdFTPP. References (1) D. Badocco, A. Mondin, P. Pastore, S. Voltolina, S. Gross, Anal. Chim. Acta 627 (2008) 239. (2) C. Chu, Y. Lo, Sensors and Actuators B. Chemical 134 (2008) 711. (3) D. Badocco, P. Pastore, Anal. Chem. 80 (2008) 2091.

SISTEMA OTTICO PER LA MISURA SIMULTANEA DI PARAMETR I BIOCHIMICI: APPLICAZIONE ALLA DETERMINAZIONE DELLA SEPSI

Francesco Baldini 1, Ambra Giannetti 1, Folco Senesi 1, Cosimo Trono 1

Luca Bolzoni 2, Giampiero Porro 2

1 Istituto di Fisica Applicata “Nello Carrara” (IFAC-CNR) Via Madonna del Piano 10, Sesto Fiorentino (Firenze), [email protected]

2 Datamed s.r.l, Via Papa Giovanni XXIII 45, Rodano (Milano)

Un’esigenza, manifestatasi nel settore biomedico nell’ultimo decennio e assurta negli ultimi anni a requisito spesso essenziale, è rappresentata dalla necessità di ottenere l’analisi di campioni biologici in tempi brevi e tramite una strumentazione vicina al paziente, senza dover ricorrere a laboratori di analisi con conseguenti ritardi nella diagnosi o nella definizione della corretta terapia. E’ infatti evidente come in certe particolari patologie o in pazienti a rischio, la rapidità nella diagnosi e nella scelta della terapia costituiscano aspetti determinanti per la vita del paziente. In questo ambito, è stata realizzata una nuova piattaforma ottica funzionante per fluorescenza per la misura di parametri biochimici, compatta e potenzialmente adatta per applicazioni vicino al letto del paziente. Tale dispositivo è stato caratterizzato tramite due saggi di tipo sandwich, per la determinazione della proteina C reattiva (CRP) e della procalcitonina (PCT), parametri essenziali per una corretta diagnosi della sepsi 1,2. Il cuore della piattaforma è un chip in polimetilmetacrilato (PMMA), contenente 13 microcanali (altezza 50 µm, larghezza 600 µm e lunghezza 10 mm), attraverso cui fluisce il campione esaminato. Lo strato sensibile è immobilizzato sulla parete superiore di ciascun microcanale. La fluorescenza, che si genera in prossimità dell’interfaccia campione/plastica, viene in gran parte emessa verso la plastica e, grazie ad una particolare sagomatura del chip, viene guidata parallelamente a ciascun microcanale 3. Un diodo laser eccita perpendicolarmente lo strato sensibile e la fluorescenza emessa viene raccolta lateralmente da una fibra ottica e inviata ad un fotodiodo amplificato, dopo un opportuno filtraggio ottico che rimuove ogni contributo proveniente dalla sorgente. Una slitta motorizzata permette una scansione automatica dei 13 microcanali. La piattaforma è anche dotata di una fluidica che permette il trattamento del campione in termini di diluizione e incubazione. Lo strato sensibile è costituito dall’anticorpo di cattura immobilizzato covalentemente sul PMMA (clone C5 per la CRP e clone PROC1 3G3 per la PCT). Le condizioni per i due saggi sono: i) incubazione di 10 minuti dell’antigene; ii) lavaggio per tre minuti, iii) incubazione di 10 minuti con l’anticorpo secondario (clone C7 per la CRP e clone PROC4 6C6 per la PCT) marcato con il fluoroforo DY647; iv) lavaggio per tre minuti. La velocità di flusso utilizzata in tutti i passaggi è di 10 µL min-1. In Figura 1 sono mostrate le curve di calibrazione per la CRP e la PCT, mentre la Tabella 1 riassume le prestazioni dei due saggi 4,5. Riferimenti 1. P. M. Ridker, J. E. Buring, N. R. Cook, N. R. Rifai, Circulation 107 (2003) 363. 2. W. Weglohner, J. Struck, C. Fischer-Schulz, N. G. Morgenthaler, A. Otto, C. Bohuon, A. Bergmann,

Peptides 22 (2001) 2099. 3. F. Baldini, A. Carloni, A. Giannetti, G. Porro, C. Trono, Anal. Bioanal. Chem. 391 (2008) 1837. 4. F. Baldini, A. Carloni, A. Giannetti, G. Porro, C. Trono, Sensors and Actuators B 139 (2009) 64. 5. F. Baldini, L. Bolzoni, A. Giannetti, M. Kess, P. M. Krämer, E. Kremmer, G. Porro, F. Senesi, C. Trono,

Anal. Bioanal. Chem. 393 (2009) 1183.

Figura 1. Curve di calibrazione per la CRP (in alto) e la PCT (in basso).

Analita Intervallo di funzionamento (mg L-1)

Limite di rivelazione

CRP 0.1-300 5 µg L-1

PCT 1 - 1000 12 µg L-1

Tabella 1. Prestazioni ottenute per i due saggi per la CRP e la PCT.

TOWARDS A BIOLUMINESCENCE-BASED MULTIPLEXED BIOSENS ING PLATFORM FOR ON FIELD MONITORING OF XENOBIOTICS AND ENDOCRIN E DISRUPTORS

Luca Cevenini, Elisa Michelini, Laura Mezzanotte, Aldo Roda

Dept. of Pharmaceutical Sciences, Via Belmeloro 6, University of Bologna

[email protected] The on field monitoring of the food and the environment for health threatening agents is a difficult task since available techniques usually require pre-analytical and purification steps and sophisticated equipment. Alternatively, unconventional analytical tools that consent a rapid and low cost monitoring of complex matrices (e.g., water, food, soil) are provided by bioluminescent (BL) whole-cell biosensors. Whole-cell sensing systems measure a cell's response to a specific analyte, or a class of analytes.1 The response, which may include activation or inhibition of signal transduction pathways, is then converted into the expression of a luminescent reporter protein (e.g., luciferase, green fluorescent protein), giving a specific signal proportional to the amount of analyte present in the sample. The present work describes the development of a new portable biodevice containing engineered bioluminescent whole-cell biosensors2 for different analytes. Genetically engineered cell-based sensing systems were obtained by introducing into bacterial yeast or mammalian cells a suitable vector expressing a reporter protein, e.g., a luciferase, whose expression is controlled by regulatory proteins and promoter (O/P) sequences. The regulatory protein is able to recognize the presence of the analyte (e.g., compounds with hormone-like activity, heavy metals…) and to consequently activate the expression of the reporter protein, by unbinding the O/P region. The reporter protein can then be readily measured and directly related to the analyte concentration in the sample. Different recombinant yeast and bacterial biosensors were immobilized in a modified clear bottom black 384 well microtiter plate to obtain a bioluminescent cell array that can be stored for up to 1 month at 4°C without loosing cell vitality. 3 For example dose-response curves for testosterone obtained with the yeast biosensor for androgens after 2 weeks and 1 month storage of immobilized cells kept at 4°C showed a LOD of 0.1 nM and 0.5 nM for testosterone, respectively. The cell array is in contact, through an optical taper, with an imaging light sensor, a portable charge-coupled device (CCD) camera able to localize and quantify the luminescent signal. “Contact” light detection, in which the signal is produced on (or very close to) the detection surface, allows to achieve a much higher optical efficiency than that of conventional camera-based imaging systems. The applicability of this multiplexed biosensing platform containing whole-cell biosensors to measure hormonal activity in environmental and food samples is reported. References 1. E. Michelini, M. Magliulo, P. Leskinen, M. Virta, M. Karp, A.Roda. Clin Chem. 51 (2005) 1995-8E. 2. L. Michelini, L. Cevenini, P. Mezzanotte, M. Leskinen, M. Virta, A. Karp, Roda, Nat Protoc 3 (2008) 1895. 3. E. Michelini, A. Roda, L.S. Dolci, L. Mezzanotte, L. Cevenini. Patent RM2009/A000064.

AMINOACIDS AND OLIGOPEPTIDES AS USEFUL TOOLS FOR BI OSENSING PURPOSES IN FOOD ANALYSIS

Michele Del Carlo, Marcello Mascini, Manuel Sergi, Dario Compagnone

Dipartimento di Scienze degli Alimenti, Università di Teramo, via C..R. Lerici 1, [email protected]

Biosensing technology is predicted to have an impact on food safety and quality if the developed methods and devices will be able to produce extensive data for anticipatory studies (i.e., emerging or new pathogens), regulatory (i.e., general toxicity index associated to group of contaminants), or historical series (i.e., geographical area of risk). In this respect the search for new bioreceptors to be incorporated in the sensing system has led to the production of biological recognition elements (mainly antibodies and aptamers) having high affinity and selectivity for the target analytes (i.e toxins, antibiotics). These have been implemented for multiplexing in optical or electrochemical array formats. The major drawback of this approach is the effect of complex food matrices on the biological molecule; pre-treatment of the sample for purification and pre-concentration is often required and a careful calibration for accurate quantitative data output is needed. In this presentation data on the exploitation of the use of short aminoacidic sequences as analytical tool will be presented. These bioreceptors have lower affinity and selectivity then antibodies selectivity presenting advantages in terms of cost, ease of preparation, robustness and regeneration of the bioreceptor. Sequences obtained merging literature data information and rational design have been used for solid phase extraction of the target analytes or coupled to different transducers for the realisation of biomimetic biosensors (1-2). Examples of oligopeptides used as solid phase extraction matrix for organophosphate and carbamate pesticides and for Ochratoxin A will be illustrated (3-4) This SPE supports can be easily regenerated for 50-80 assays and are cost effective vs the immunoaffinity approach. Both systems have been coupled to electrochemical detection strategies for the development screening assays. More recently, aminoacidic units have been incorporated in conducting polymers. These are attractive transduction materials for sensor applications, since they are able to convert a molecular binding event into a measurable electrical or optical signal. We have realized aminoacid-modified poly(para-arylene ethynylene)s (PAEs) using an innovative synthetic approach. Functionalisation of monomers and polymers with aminoacidic arms has led to the production of molecules very selective for the detection of Hg(II) (5). Data on the use of functionalised PAEs films for the realisation of optical sensing platforms will be presented. References

1. M. Mascini, A. Macagnano, G. Scortichini , M. Del Carlo, G. Diletti, A. D’Amico, C. Di Natale, D. Compagnone, Sensors and Actuators B 111-112 (2005) 376.

2. M. Mascini, M. Del Carlo, D. Compagnone, I. Cozzani, P.G. Tiscar, C.P. Mpamhanga, B. Chen, Analytical Letters 39 (2006) 1.

3. M. Mascini, M. Sergi, D. Monti, M. Del Carlo, D. Compagnone, Analytical Chemistry 80 (2008) 9150. 4. M. Di Gregorio, A. Visconti, M. Pascale, D. Compagnone, Journal of Agricultural and Food

Chemistry, in press 5. D. Compagnone, A. Ricci, M. Del Carlo, L. Prodi, S. Bonacchi, D. Viallamaina, M. Chiarini, M.T.

Apicella, C. Lo Sterzo, Luminescence 23 (2008) 212.

SUPRAMOLECULAR SENSING WITH CAVITANDS

Enrico Dalcanale

Dipartimento di Chimica Organica e Industriale, Università di Parma, Parco Area delle Scienze 17/A, 43100 Parma, Italy

[email protected] The quest for selectivity is one of the key issues involved in developing new, efficient chemical sensors.1 The use of supramolecular structures has proven to be one of the best approaches to generate new materials with molecular specificity for chemical sensing.2 However, the direct translation of the molecular recognition properties of a given receptor from solution studies to the solid devices is not trivial, since non specific interactions such as dispersion forces, and material properties, like surface morphology, come into play.3 The solution to the problem relies in mastering the weak interactions responsible of molecular recognition at the solid-gas interface. In this contribution two examples will be illustrated: • Cavitands as selective layers for mass/fluorescence sensors to illustrate the basic science4 • A portable sub-ppb level monitoring system for aromatic volatile organic compounds (VOC)

(Figure 1).5 In the first example the focus will be on the design of receptors capable of synergistic multiple interactions. Comparison of the responses of the different cavitands toward linear alcohols shows that increasing incrementally the number of cooperative interactions leads to higher selectivity (the entropic approach). In the second example a practical application will be highlighted. The recognition element is not integral part of the sensor, but it operates before the sensor element, providing the desired analyte in pure form for highly sensitive environmental detection. A miniaturized system is proposed, composed of a selective supramolecular concentration unit, a Si-micromachined GC column and a Si-integrated Metal Oxide Semiconductor sensor (MOX). The issue of achieving at the same time molecular-level selectivity and low-ppb sensitivity for benzene has been solved by disconnecting the recognition element from the detection unit. The recognition event is assigned to a molecular receptor, capable of selectively trapping aromatic vapors at the gas-solid interface. The selective concentration component is interfaced to a miniaturized Si GC column, necessary for the separation of the different aromatic compounds released by the trapping unit, which are then individually channeled to the MOX detector.

Figure 1

References 1. L. Pirondini, E. Dalcanale, Chem. Soc. Rev. 36 (2007) 695. 2. R. Pinalli, M. Suman, E. Dalcanale, Eur. J. Org. Chem. (2004) 451. 3. M. Tonezzer, M. Melegari, G. Maggioni, R. Milan, G. Della Mea, E. Dalcanale, Chem. Mater. 20 (2008)

6535. 4. M. Melegari, M. Suman, L. Pirondini, D. Moiani, C. Massera, F. Ugozzoli, E. Kalenius, P. Vainiotalo, J.-C.

Mulatier, J.-P. Dutasta, E. Dalcanale, Chem. Eur. J. 14 (2008) 5772. 5. S. Zampolli, P. Betti, I. Elmi, E. Dalcanale, Chem. Commun. (2007) 2790.

INFLUENCE OF THE REAL BACKGROUND SIGNAL ON THE LINE ARITY OF THE STERN-VOLMER CALIBRATION FOR THE DETERMINATION OF M OLECULAR

OXYGEN WITH OPTICAL SENSORS

Denis Badocco, Andrea Mondin, Alberto Fusar1 and Paolo Pastore

1Dipartimento di Scienze Chimiche, Università di Padova, v. Marzolo 1 [email protected]

The role of light emission components unrelated to the Stern-Volmer kinetic model (SV), in the curvature of the Stern-Volmer model (SV) was evidenced and the discrepancy often encountered between calibrations with light intensity, I, and luminescence lifetime,τ , was explained by means of mathematical models applied to experimental data.1 Sensing membranes prepared embedding tris-4,7-diphenyl-1,10-phenanthroline (Ru(dpp), platinum(II) meso-tetra-phenyl porphyrin (PtTPP) and palladium(II) meso tetra (pentafluoro-phenyl) porphyrin, (PdFTPP) in a polysulfone matrix, were used to confirm the proposed theory2. Multisite hypotheses or other correction procedures3 must be invoked only after a careful evaluation of the background light really present in the system. A new form of the SV equation having the advantage to obtain the '

SVK and the effective

background with a simple un-weighed regression method was proposed and compare to the “classical” SV requiring the knowledge of the background and a weighed regression. Experimental results confirmed the theory for what concerns the concentration limit for the determination of oxygen which may be theoretically determined. Ru(dpp)OS, PtTPP and PdFTPP membranes may determine oxygen mixtures up to 100, 60.7 and 19.0 %, respectively, with a resolution of 2%. Narrower working intervals are explorable if better resolution is required.

0 25 50 75 1000

2

4

6

0

2

4

6a

τ 0/τ-1

I 0/I-1

%O2

PdFTPP

PtTPP

Ru(dpp)OS

0 20 40 60 80 1000

2

4

6

0

2

4

6

τ 0/τ-1

I 0/I-

1

%O2

PdFTPP

PtTPP

Ru(dpp)OS

b

Figure 1. a) SV calibration plots of Ru(dpp)OS, PtTPP and PdFTPP membranes in terms of I (uncorrected for real background, empty symbols) and τ (from mono-exponential fitting, black symbols). b) SV calibration plots in terms of I (empty symbols)and τ (black symbols) correct of real background. References (1) L. Huynh, Z. Wang, J. Yang, V. Stoeva, A. Lough, I. Manners, M.A. Winnik, Chemistry of Materials 17

(2005) 4765. (2) M. Biesaga, K. Pyrzynska, M Trojanowicz, Talanta, 51 (2000) 209. (3) S. Draxler, M. E. Lippitsch, I. Klimant, H. Kraus, O. S. Wolfbeis, J. Phys. Chem. 99 (1995) 3162.

DETERMINAZIONE DEL As(III) MEDIANTE VOLTAMMETRIA DI STRIPPING ANODICO CON UN SENSORE D’ORO LATERALE

Ornella Abollino, Agnese Giacomino, Mery Malandrino, Edoardo Mentasti

Dipartimento di Chimica Analitica, Università di Torino, Via Pietro Giuria 5,

[email protected]

L’avvelenamento da arsenico può causare diversi disturbi, tra cui malattie epidermiche, respiratorie, cardiovascolari, gastrointestinali; può avere inoltre effetti cancerogeni e mutageni. Nel corso degli anni sono state registrate diverse e spesso consistenti contaminazioni da arsenico di falde acquifere e terreni in varie parti del mondo, come in Stati Uniti e Regno Unito, e soprattutto in Bangladesh, Cina e India. Recentemente, anche l’Unione Europea ha ridotto il limite nelle acque potabili da 10 a 5 µg/l. Da queste considerazioni nasce la necessità di poter disporre di tecniche per la determinazione dell’arsenico e, date le basse quantità d’interesse, di tecniche analitiche sufficientemente sensibili; inoltre la differenza in tossicità delle varie specie evidenzia l’interesse di poter disporre di un metodo che permetta di poter distinguere il contributo di As(III) e As(V). I metodi riportati in letteratura per la determinazione e speciazione dell'arsenico sono generalmente metodi spettroscopici. Queste metodiche però richiedono strumenti costosi, procedure lunghe e complicate, pretrattamenti particolari del campione, e non sono esenti da problematiche di interferenza. Inoltre, generalmente non sono in grado di effettuare direttamente un’analisi di speciazione, ma richiedono di essere accoppiate a tecniche separative. I metodi elettrochimici potrebbero rappresentare un’interessante alternativa in quanto sono in grado di distinguere i diversi stati di ossidazione, hanno bassi costi, sono molto sensibili, rapidi e versatili. Per questo motivo, negli anni sono state proposte diverse metodologie voltammetriche per la determinazione dell’arsenico. Scopo di questo lavoro è stata la messa a punto e l’ottimizzazione di una procedura per la determinazione dell’arsenico a livello di traccia ed ultratraccia mediante voltammetria di stripping anodico con sensore d’oro laterale1. Sono stati presi in esame diversi elettroliti di supporto e diverse tipologie di scansione di potenziale (differenziale ad impulsi ed onde quadre). È stato valutato l’effetto di alcuni parametri elettrochimici sul segnale voltammetrico di 5 µg/l di Hg. L’elettrolita di supporto e la scansione che hanno permesso di ottenere i migliori risultati sono stati HCl 0.25 M e la scansione differenziale ad impulsi. I valori ottimali sono risultati essere: step potential: 0.018 V; interval time 0.1 s; modulation amplitude 0.15 V. Sono poi stati valutati l’effetto del potenziale di deposizione e del tempo di deposizione sul segnale analitico. Il segnale più intenso e la migliore linea di fondo sono stati ottenuti utilizzando -0.30 V come potenziale di deposizione. Sono state quindi valutate linearità, accuratezza, ripetibilità, limiti di quantificazione e di rivelabilità, e l’effetto della presenza di potenziali interferenti anionici e cationici. Si è infine valutata la possibilità di applicare il metodo al monitoraggio di acque potabili. È stato inoltre studiato il comportamento elettrochimico dell'arsenico(III) all'elettrodo attraverso la voltammetria ciclica, e si sono confrontati i dati sperimentali con quelli teorici ottenuti attraverso il calcolo. Parallelamente, è stato effettuato uno studio delle caratteristiche e dei cambiamenti nel tempo della superficie d’oro del sensore per mezzo di voltammetria ciclica, microscopia ottica e microscopia a scansione elettronica. Riferimenti 1. X. Dai, O. Nekrassova, M.E. Hyde, R.G. Compton, Analytical Chemisty 76 (2004) 5924. 2. R. Feeney, S.P.Kounaves, Talanta 58 (2002) 28.

DEVELOPMENT OF AMPEROMETRIC IMMUNOSENSORS BASED ON NANOBIOCOMPOSITE MATERIALS

Marco Giannetto, Maria Careri, Lisa Elviri, Alessandro Mangia, Giovanni Mori, Francesca Speroni

Dipartimento di Chimica Generale ed Inorganica, Chimica Analitica, Chimica Fisica, Università di Parma, Viale G.P. Usberti, I-43124 Parma, Italy

[email protected]

A recent study concerning the development of new amperometric immunosensors based on the working principle of the ELISA assays is presented. The peculiar properties of nanobiocomposite materials based on gold nanoparticles were exploited for the immobilization of Antibody/Antigen/Antibody-HRP sandwich on the glassy carbon electrode surface. HRP PA HRP PA HRP PA HRP

Figure 1. Schematic representation of the “sandwich” immobilized on the GC electrode surface.

The electrochemical transduction is mediated by thionine. The activity of such a mediator is fundamental for the electron transfer from the electrodic surface to the active redox center of HRP. Thionine was tested both in solution and after immobilizing it through electropolymerization. The immobilization protocol was applied successfully to different biological systems, the assays requiring very small amounts of sample (10 µl). Interesting findings of the developed sensors indicate that their sensitivity and response range are strongly depending from the size of the nanostructured gold substrate. Gold nanoparticles were in fact deposed on the GC surface by a suitable electrodeposition procedure that allowed to effectively control their size. Best results, in terms of homogeneity, evaluated by means of SEM analyses, reproducibility of the nanostructured layer and sensitivity were obtained with 100 nm sized nanogold obtained by double potential step electrolysis Other redox mediators (aminophenols) are also being studied in order to assess possible effects on the sensitivity of the assays. Studies aimed to the amplification of the response by means of dendrimer-based linking of the primary antibodies have also been undertaken. The developed immunosensors could be exploited as powerful tools for screening analyses in clinical and forensic fields.

SVILUPPO DI UN SENSORE POTENZIOMETRICO PER LA VITA MINA C

Domenica Tonelli, Marco Giorgetti, Adriana Mignani, Lorella Guadagnini, Erika Scavetta

1 Dipartimento di Chimica Fisica ed Inorganica, Università di Bologna, Viale Risorgimento, 4 40136 BOLOGNA, [email protected]

Questo contributo descrive lo sviluppo di un sensore potenziometrico per l’acido ascorbico, basato sulla modifica di elettrodi di carbone vetroso (GC) con film di polipirrolo a stampo molecolare (MIP-polyPy), elettrosintetizzati in diverse condizioni sperimentali [1]. Vengono confrontate le prestazioni dei film stampati con quelle dei polimeri depositati in assenza di ascorbato (NIP-polyPy), in condizioni in cui essi sono ancora conduttori o quando sono stati sovraossidati. I primi risultati ottenuti mostrano che gli elettrodi a base di MIP-polyPy sovraossidato presentano le migliori prestazioni sia in termini di pendenza del responso potenziometrico, di ampiezza dell’intervallo di risposta, che di selettività (figura 1).

-5.5 -5.0 -4.5 -4.0 -3.5 -3.0 -2.5-20

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Figura 1. Curva di risposta potenziometrica ottenuta per l’elettrodo MIP-polyPy sovraossidato in cui è stata rimossa l’impronta molecolare, in tampone fosfato, pH 5.0.

La selettività è stata determinata con il metodo del Matched Potential [2] ed è risultata particolarmente soddisfacente. Infatti si può affermare che i soli anioni, tra quelli analizzati, che potrebbero interferire nella determinazione dell’acido ascorbico, risultano il citrato, l’acetilsalicilato e il bicarbonato. L’elettrodo ISE è stato applicato per determinare la vitamina C in campioni reali di succhi di arancia e di un prodotto farmaceutico, in forma di compressa. Il solo trattamento a cui sono stati sottoposti i campioni era la solubilizzazione in tampone fosfato. I risultati ottenuti sono abbastanza in accordo con quanto dichiarato sul bugiardino o sulla confezione del succo di frutta, se valutati con il metodo dell’aggiunta standard, anche se la concentrazione risultante per il prodotto farmaceutico risulta leggermente in eccesso (+9%), in quanto la matrice contiene una notevole quantità di sostanze interferenti. Il limite principale del sensore è costituito dal tempo di risposta e di “stabilizzazione” della membrana polimerica che, attualmente, è dell’ordine di circa 10 minuti. Sono in corso studi per valutare se modificando lo spessore della membrana tale parametro possa diminuire in modo sensibile. Riferimenti 1. L. Ozcan, M. Sahin, Y. Sahin, Sensors 8 (2008) 5792. 2. Y. Umezawa, K. Umezawa, H. Sato, Pure Appl. Chem. 67 (1995) 507.

DEVELOPMENT OF PIEZOELECTRIC- AND SPR IMAGING-BASED BIOSENSORS FOR GENE DOPING DETECTION

S. Scarano1, M. Spiriti2, P. Bogani2, M. Buiatti2, M. Mascini and M. Minunni

1Chemistry Department, Univ. of Florence, v. della Lastruccia 3, S., Sesto F.no, 50019, Florence

2Evolutionary Biology, Leo Pardi, Via Romana 17, 50125 Firenze [email protected]

The sport and anti-doping authorities fear that the newer form of doping, so-called gene doping, based on a misuse of gene therapy, will be undetectable and thus much less preventable. Gene doping represents an open challenge for new analytical protocols, because target analytes, sampling and detection strategies are far from being optimized. In this context, biosensors are proving to be a promising approach to this aim. In this work we aimed at developing label free DNA-based biosensors, based on Quartz Crystal Microbalances (QCM) and on SPR imaging (SPRi) for detecting target sequences used as transgenosis markers in animal and human cells, in line with our previous work for Genetically Modified Organism detection (GMOs) in different matrices of plant origin, using suitable markers. In particular, Enhanced Green Fluorescent Protein (EGFP) gene and Cytomegalovirus (CMV) promoter sequences, used as model systems, were chosen as proof of principle. Both QCM and SPRi systems were optimized using synthetic oligonucleotides and then applied to the detection of the EGFP reporter gene and of CMV promoter sequences in PCR products obtained from pEGFP-C1 purified plasmid. The presence of human genomic DNA was evaluated for possible matrix effect and, finally, the detection of these transgenosis markers has been achieved by piezoelectric system in DNA extracted and amplified from human cell culture (HEK i.e. Human Embryonic Cells) transformed with the same plasmid, demonstrating the suitability of biosensor approach for transgene detection in samples of human origin. Acknowledgements: We thank Prof. A. Arcangeli, Dip. di Patologia ed oncologia sperimentale, Università degli Studi di Firenze for kindly providing human transgenic cells (HEK-EGFP and HEK-293) and the Word Antidoping Agency (WADA), for finding the project “Affinity-Based Biosensing (ABBs) for gene doping detection: an integrated approach” within the “Scientific Research Grant 2008,Competition -topic C. Detection of Prohibited Substances/Methods: novel methodologies”, Coordinator: M. Minunni.

INFLUENCE OF POLYMERIC MATRIX ON OPTICAL OXYGEN SENSOR PERFORMANCE

Denis Badocco, Andrea Mondin1, Alberto Fusar and Paolo Pastore

1 Department of Chemical Sciences, University of Padua, Via Marzolo 1, 35131 Padua, Italy

[email protected] The determination of oxygen concentration is important in many areas of industry, medicine and environment. Oxygen optical sensors are more and more attractive than conventional amperometric devices, because, in general, they have a faster response time, they do not consume oxygen and are less easily poisonable. Thin films containing oxygen-sensitive luminophores are widely used as in pressure sensitive paints (PSPs) because of their high sensitivity. Sensor operation is based on the quenching of luminescence in the presence of oxygen. Luminophores are commonly embedded in a polymeric matrix. Following the Stern-Volmer (SV) model, film sensibility ( '

SVK ) is proportional to its permeability toward oxygen. The polymer

may also influence the lifetime of the luminophore because it may modify the efficiency of non radiative processes due to luminophore-polymer interaction. Some matrices can also generate deviations from the linearity of the SV model, if they cannot be considered as a homogeneous media. If this happens a multisite model must be applied. The matrix may also enhance or diminish degradation of the embedded metal-complex due to light dependent phenomena and ageing processes, such as solvent residual evaporation and pore collapse. The aim of the present work is to show the results of some recent activities of our research group on different oxygen sensing membranes containing Platinum-tetraphenylporphyrin (PtTPP) embedded in various polymers. Each membrane was characterized for its optical properties and tested using the real background determination and various calibration models. A strong variation (100 times) of sensibility by varying the polymer nature was determined. Accuracy of each membrane was determined and rationalized using a numeric simulation1. A sensor containing 2 luminophores was created using suitable polymer and luminophores to obtain maximum accuracy and sensibility over the range 0-100% O2. Figure 1 a,b shows typical calibration plots (a) and the emission behavior (b).

0 10 20 30 40 500

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Figure 1 . a) SV plot for 4 membranes containing PtTPP embedded in different polymers: PS: polystyrene; EtC Ethylcellulose; PSF: polysulfone; PVC: polyvinylchloride b) Normalized emission profiles %O2–100 %N2 vs. time (profiles were corrected for background) %O2 = 0.5, 1, 1.5, 2.5, 3.5, 5, 7.5, 10, 15, 20, 30, 40, 50 %O2

References 1. D.Badocco, P.Pastore, Anal.Chem. 80 (2008) 2091.

POLYACRYLIC MOLECULARLY IMPRINTED POLYMERIC MEMBRAN ES AS RECEPTORS FOR ELECTROCHEMICAL SENSORS

Girolamo D’Agostino, Maria Pesavento, Antonella Profumo, Valeria Vacchelli

Dipartimento di Chimica Generale, Università di Pavia,Via Taramelli 12. 27100-Pavia-Italy [email protected]

Molecular Imprinted Polymers (MIP) are synthetic polymers containing sites able to selectively recombine the template molecule. For this reason they are widely used as specific adsorbents. In addition since the pioneering works of Mosbach, Piletsky, and others1, 2, 3, MIPs are employed as recognition elements in electrochemical sensors. Potentiometric transduction has been found to be well suited for detection of positively charged templates4. Voltammetry3 and amperometry5 have been used too. In this case the electrical resistance of the polymeric layer must not be too high. This has been faced by different approaches, for example using conducting polymers, very thin layers, even monolayers, or by using porous gels with ionic conduction. MIP membranes are useful for the selective preconcentration of the template at the electrode, and so could contribute to improve the detection limit. To this aim, the presence of strong and specific sites for the recombination of the template is important. In this investigation acrylic monomers have been used to prepare homogeneous thick membranes, directly in contact with the electrochemical transduction device. The imprinting method was similar to that previously used4 with methacrylic acid (MAA) as functional monomer and ethylene glycol dimethacrylate (EGDMA) as cross-linker was. In some cases EGDMA was used both as cross-linker and functional monomer (MIP-CL). Different molar ratios of functional monomer to crosslinker were studied. The current at electrodes modified with different polyacrylic polymers, imprinted (MIP and MIP-CL) and non imprinted (NIP and NIP-CL), was studied by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Cyclic voltammograms obtained with screen printed graphite electrodes (SPGE) modified with a thick layer of acrylic polymers are reported for example in the Figure.

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Figure : CV of MIP (A) and MIP–CL (B) on SPGE in HClO4 0.03M. MIP is for TNT.

The presence of carboxylic groups produces a non faradic current similar to that in the absence of carboxylic groups, while the current at -1V (vs Ag/AgCl) is much higher in the presence of carboxylic group, and in NIP (non imprinted polymers). CV were obtained in 0.03 M sodium perchlorate at pH=6.5 too. They were similar to those at acidic pH, but the current was lower in the less acidic solution. Highly mobile ions, as protons, produce an higher current than sodium. The recombination with an electroactive species, as TNT, is clearly seen by DPV on both MIP and MIP-CL, with reduction peaks at around -0.4V and -0.6V (vs Ag/AgCl) for MIP and bare electrode at pH 1.5. The peaks are located at more cathodic potential at higher pH. The selectivity of MIP electrodes was good, better than that of the MIP-CL electrodes. NIP modified electrodes too gave a signal in the presence of the template, but lower than that of MIP, and not selective for other nitroderivates. Not any DPV signal was registered with NIP-CL, while a current flowed. References

1. G. Vlatakis, L.I. Andersson, R. Müller, K. Mosbach, Nature (London) 361(1993) 645. 2. S.A. Piletsky, A.P.F. Turner, Electroanalysis 14 (2002) 317. 3. C. Blanco-Lopez, M.J. Lobo-Castanon, A.J. Miranda-Ordieres, P.Tunon-Blanco, Trends Anal. Chem.

23 (2004) 36. 4. G. D’Agostino, G. Alberti, R. Biesuz, M. Pesavento, Biosensors and Bioelectronics 22 (2006) 145. 5. A. Ramanaviciene, A. Ramanavicius, Biosensors and Bioelectronics 20 (2004) 1076.

A B

COLLISIONAL MECHANISM BASED E-DNA SENSORS: A GENERAL PLATFORM FOR LABEL-FREE ELECTROCHEMICAL D ETECTION OF

DNA BINDING PROTEINS AND PROTEIN/SMALL MOLECULE INT ERACTIONS

Francesco Ricci1, Kevin J. Cash2, , Kevin W. Plaxco3,4, Giuseppe Palleschi1

1Dipartimento di Scienze e Tecnologie Chimiche, Universita’ di Roma, Tor Vergata, Roma. 2Department of Chemical Engineering, 3Department of Chemistry and Biochemistry, 4Program in

Biomolecular Science and Engineering, University of California, Santa Barbara. [email protected]

Here we demonstrate a reagentless, electrochemical platform for the specific detection of proteins that bind to single- or double-stranded DNA1. The sensor is comprised of a double- or single-stranded, redox-tagged DNA probe which is covalently attached to an interrogating electrode. Upon protein binding the current arising from the redox tag is suppressed, indicating the presence of the target. Using this approach we have fabricated sensors against the double-stranded DNA binding proteins TATA-box binding protein and M.HhaI methyltransferase, and against the single-strand binding proteins E. coli SSBP and replication protein A. All four targets are detected at nanomolar concentrations, in minutes, and in a convenient, general, readily reusable, electrochemical format. The approach is specific; we observed no significant cross-reactivity between the sensors. Likewise the approach is selective; it supports, for example, the detection of single strand binding protein directly in crude nuclear extracts. The generality of our approach (including its ability to detect both double- and single-strand binding proteins) supports a collisional signaling mechanism in which binding alters the collision efficiency, and thus electron transfer efficiency, of the attached redox tag. We have demonstrated this hypothesis designing a general, sensitive and selective approach for the detection of proteins binding to specific small molecules2. The electrochemical approach is based on a redox-tagged DNA signaling scaffold conjugated to a small molecule receptor and covalently attached to an interrogating electrode. In the absence of target, this scaffold is relatively dynamic, allowing collisions between the redox tag and electrode surface and supporting relatively efficient electron transfer. The binding of a protein to the small molecule receptor alters the dynamics of the scaffold, minimizing collisions and thus reducing the observed current. We optimized the scaffold using a biotin receptor to determine optimal structure before then applying it to detection of anti-digoxigenin antibodies using the steroid drug as the receptor. Both sensors are sensitive (detection limits in the low nanomolar), rapid and selective enough to function directly in complex matrices, including blood serum, soil suspensions and foodstuffs. References 1. K.J. Cash, F. Ricci, K.W. Plaxco, J. Am. Chem. Soc. 131 (2009) 6955. 2. F. Ricci, A.J. Bonham, A.C. Mason, N.O. Reich, K.W. Plaxco, Anal. Chem. 81 (2009) 1608.

DEVELOPMENT OF AN INTEGRATED POINT-OF-CARE TESTING DEVICE INCLUDING A PRE-ANALYTICAL SAMPLE TREATMENT SYSTEM

BASED ON FIELD-FLOW FRACTIONATION

Barbara Roda1, Mara Mirasoli2, Luca Cevenini2, Sonia Casolari1, Pierluigi Reschiglian1, Aldo Roda2

1Department of Chemistry, University of Bologna, Via Selmi 2, 40126 Bologna [email protected]

2Department of Pharmaceutical Sciences, University of Bologna, via Belmeloro 6, 40126 Bologna The “Point-Of-Care Testing" (POCT) approach is based on the development of portable analytical platforms suitable to perform analysis directly in any required place, through the use of simple analytical tools addressed to operators with limited technical skills. A POCT device should combine portability, minimum sample pre-treatment and allow performing highly sensitive multiplexed assays in a short assay time. Microfluidic integrated systems relying on biospecific recognition reactions (e.g., immunological reactions, nucleic acids hybridization) and ultrasensitive bio-chemiluminescence detection techniques represent one of the most promising options. To enable their use outside of a well-controlled environment like a central-traditional laboratory, POCT devices should also comprise an integrated module for on-line sample pre-analytical treatment and/or clean-up to achieve high sensitivity in complex matrices. With this respect, field-flow fractionation (FFF) techniques, a family of flow-assisted separation techniques which can separate analytes based on their morphologic characteristics (size, shape and superficial properties) can be exploited to develop pre-analytical modules for cells or macromolecules (e.g., proteins, protein complexes or adducts) fractionation, thus providing a selectively enriched fraction for the analysis with an increase of overall analytical output. In particular, gravitational FFF (GrFFF), exploiting the Earth gravitational field to structure the separation, appears to be particularly suited for integration in POCT devices, thanks to the simplicity of its separative device, amenable to miniaturization1. To obtain compact GrFFF devices for POCT we studied the influence of an original curvilinear geometry and miniaturization of the channel on fluidic conditions and fractionation parameters. These new channel designs resulted particularly adapt to the development of compact GrFFF tools to be easily integrated in more complex and specific analytical systems with maintenance of separative properties such as soft fractionation mechanism, simplicity, high sample recovery and selectivity. In addition, to increase GrFFF separation efficiency, a hybrid immunomodulated GrFFF system was realized by functionalizing the accumulation wall of a GrFFF channel with specific antibodies2. The final aim of this work is the development of a GrFFF-assisted module to be integrated within a lab-on-a-chip microfluidics-based device, relying on biospecific recognition reactions combined with ultrasensitive bioluminescence and chemiluminescence detection techniques, which can be realized directly into the separative system, for example by means of “contact” imaging detection employing a CCD or CMOS detector, depending on the required detectability. The GrFFF-assisted module could provide both the sample pre-analytical purification and part of the analytical device itself. For instance, it could separate plasma from cells and eventually fractionate the cells in subpopulations. The various sample fractions could be addressed to different analytical modules, and at the same time during the fractionation process the analytical information on the cellular components, which is related to biophysical features of the cells such as size, shape, density, rigidity, could be obtained. References 1. B. Roda, A. Zattoni, P. Reschiglian, M.H. Moon, M. Mirasoli, E. Michelini, A. Roda, Analytica Chimica

Acta 635 (2009) 132. 2. B. Roda, S. Casolari, P. Reschiglian, M. Mirasoli, P. Simoni, A. Roda, Analytical and Bioanalytical

Chemistry 394 (2009) 953.

UN NUOVO SENSORE AMPEROMETRICO ED OTTICO PER IL GLU COSIO

Erika Scavetta, Barbara Ballarin, Lorella Guadagnini, Adriana Mignani, Domenica Tonelli

Dipartimento di Chimica Fisica ed Inorganica, Università di Bologna, Viale Risorgimento, 4

40136 BOLOGNA, [email protected]

Questo contributo descrive lo sviluppo di un sensore amperometrico ed ottico per il glucosio, basato su un elettrodo di ITO ricoperto con l’idrotalcite (HT) Co/Al-NO3, preparata per elettrodeposizione. La deposizione elettrochimica dell’HT, basata sulla riduzione potenziostatica di una soluzione contenente cobalto e alluminio nitrato, permette di ottenere un film sottile omogeneo e meccanicamente stabile che si rivela particolarmente adatto per misure spettrofotometriche [1]. Questa HT mostra in NaOH 0.1 M un comportamento elettrocromico reversibile (Figura 1) che può essere sfruttato per lo sviluppo di un sensore ottico [2].

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Figura 1. Voltammetria ciclica, in funzione del tempo, registrata in NaOH 0.1 M a 0.005 V⋅s-1 ed assorbanza (λ =435 nm) registrata nel corso della scansione. Le foto rappresentano l’elettrodo di ITO ricoperto con il film di Co/Al HT, polarizzato a 0.0 (A) e a 0.50 V (B). Il meccanismo su cui si basa il sensore sfrutta i centri di cobalto presenti nella struttura idrotalcitica che agiscono come mediatori redox secondo lo schema seguente:

Co(III)-HT+OH-⇆Co(IV)-HT (OH-) +e- (1)

Co(IV)-HT(OH-)+glucosio � Co(III)-HT + OH-+ gluconolattone (2) L’utilizzo di un supporto elettrodico trasparente alla radiazione permette di misurare sia le variazioni di corrente che di assorbanza, in funzione della concentrazione di glucosio. Viene inoltre presentata una possibile applicazione del sensore per la determinazione del glucosio in un campione di siero bovino, sfruttando entrambe le trasduzioni. Riferimenti 1. E. Scavetta, A. Mignani, D. Prandstraller, D. Tonelli, Chem. Mat. 19 (2007) 4523. 2. E. Scavetta, B. Ballarin, M. Gazzano, D. Tonelli, Electrochim. Acta 54 (2009) 1027.

DETECTION OF DNA BY PEPTIDE NUCLEIC ACIDS AND NANOP ARTICLE-ENHANCED SURFACE PLASMON RESONANCE IMAGING

Roberta D’Agata1, Roberto Corradini2, Cristina Ferretti3, Laura Maria Zanoli4, Marcello Gatti3,

Rosangela Marchelli2, Giuseppe Spoto1,5

1Dipartimento di Scienze Chimiche, Università di Catania, Viale A. Doria 6, Catania, Italy. [email protected]

2Dipartimento di Chimica Organica ed Industriale, Università di Parma, V.le G.P. Usberti 17/a, Parma, Italy.

3Neotron S.p.a., Stradello Aggazzotti 104, S. Maria di Mugnano, Modena, Italy. 4Scuola Superiore di Catania, Università di Catania, V. S. Paolo 73, Catania, Italy.

5Istituto Biostrutture e Bioimmagini, CNR, Viale A. Doria 6, Catania, Italy.

The successful sequencing of the human genome has improved our understanding of the complex network of gene expression and has prompted towards the development of new methodologies for the simpler, faster and cheaper detection of nucleic acids to be used for both applicative and research needs in many areas including clinical diagnosis, environmental monitoring and food control. The recognition capabilities of nucleic acids by detection methods based on the hybridization reaction between an oligonucleotide (probe) and an unknown counterpart (target) are well-established. Most of the above methods require a polymerase chain reaction (PCR) amplification of the target and detect the duplex formation by using transducers or indicators able to generate a signal as a consequence of the specific hybridization event. For this purpose, various optical, electrochemical or piezoelectric sensing methods have been investigated. Unfortunately, most of the proposed assays are based on the detection of signals produced by tags which are chemically inserted into the structure of one of the hybridized strands. The labelling process may be laborious, can introduce artifacts in the detection process and may interfere with the strands hybridization. DNA sensing is expected to be significantly improved by using simple and economic detection methods which require minimal DNA modifications and provide enhanced signal amplification. In this perspective, the direct analysis of non amplified genomic DNA by using a label-free method appears an excellent cost-effective alternative that can be achieved by using ultrasensitive DNA detection tools. In this communication the results we obtained in the ultrasensitive detection of non-amplified DNA will be presented. The method is based on the use of surface-immobilized peptide nucleic acids (PNA) probes in combination with continuous-flow microfluidics and nanoparticle-enhanced surface plasmon resonance imaging (SPRI) biosensing.1-3 Riferimenti 1. R. D’Agata, R. Corradini, G. Grasso, R. Marchelli, G. Spoto, ChemBioChem 9 (2008) 2067. 2. L. Zanoli, R. D’Agata, G. Spoto, Minerva Biotec. 20 (2008) 165. 3. G. Grasso, R. D’Agata, L. Zanoli, G. Spoto, Microchemical J. (2009), doi:10.1016/j.microc.2009.05.00.

LACTOFERRIN IMMUNOSENSOR: SELECTIVITY AND NEW DEVEL OPMENTS

Mauro Tomassetti, Elisabetta Martini, Luigi Campanella

Department Chemistry of University “La Sapienza”, P.le a. Moro 5, 00185, Rome. [email protected]

Last year we fabricated a new immunosensor for the analysis of lactoferrin protein [1]. In the present research we studied the selectivity features with reference to other possible interfering milk proteins. The study was also extended to the ability of the antibody used and the analyte under test to produce the immunocomplex. As the immunoaffinity characteristic of the antigen for the immunoreagent is of the utmost importance in any immunological method, a rough estimation of the Kaff of the antigen under test (i.e. lactoferrin) was obtained by us at the midpoint of the calibration curve, where Kaff = 1/IC50. In our case IC50 was calculated as the concentration of the analyte that binds 50% of the antibody to the sensor surface. The Kaff value was found to be of the order of 1.7 x 106 M-1. The last problem tackled in the previous research [1] was the excessively long time required for each measurement, even though the measurement procedure used in the previous research was the classic “competition” ELISA type, employing an ad hoc enzymatic marker. One possibility of reducing measurement time was to perform a “direct” type of measurement. The “direct measurement immunosensors” described in literature do not involve any “competition” procedure. They usually do not make use of a marker and in these devices the signal is often obtained directly as a result of immunocomplex formation. This kind of immunosensor has generally not been very successful for various reasons even though it allows a considerable reduction in analysis time. Nevertheless we actually resumed studies on this type of immunosensor. However in this case, to perform “direct” measurement, unlike what is usually reported in the literature, an enzymatic marker was again used to perform the electrochemical measurement, while the transducer used was of the amperometric type. For the purpose of direct measurement of a real sample a completely innovative procedure was therefore developed. First of all, a one-off experimental measure was performed of the concentration of labelled antigen required to form the complex with the respective full antibody, which was immobilized on an ad hoc membrane (Immobilon). After renewing the buffer in the cell, the measurement was then performed, first of all by complexing the antigen to be measured, contained in the real sample, with the antibody immobilized in the membrane; secondly the complexation between immobilized antibody and a fixed quantity of labelled antigen was “completed” by dipping the sensor in the solution of the latter. The enzymatic measurement was then performed, after washing, by adding the specific substrate. Lastly, the signal was obtained which would be used to enter into the calibration curve by taking the difference between the signal recorded during the one-off measurement and that obtained during the measurement described above. The first application performed on real samples using the “new direct” method was the measurement of lactoferrin content in human saliva and in powdered milk. References 1. L. Campanella, E. Martini, M. Tomassetti, J. Pharm. Biomed. Anal. 48 (2008) 278.

MEMBRANE PROTEINS EMPLOYED IN OFET BIO-SENSORS

M. D. Angione1, A. Mallardi2, S. Cotrone1, G. Palazzo1 and L. Torsi1

1 Dipartimento di Chimica - Università degli Studi di Bari - Bari (Italy), [email protected] 2 Istituto per i Processi Chimico-Fisici (IPCF), CNR – Bari (Italy)

Biological assay is nowadays largely performed by high-throughput, reliable methods, such as the polymerase chain reaction (PCR) formats or the enzyme-linked immunosorbent assays (ELISA) [1]. These methods require, however, extensive sample treatment involving incubation steps, sample cleanup or amplification. The “labelling” of the bio-analyte, a pathogen such as the HIV virus for instance, is also needed to allow the transduction of the bio-recognition event, eventually adding further sample handling, before the analysis can be performed. Besides, as optical detection is used, expensive dedicated systems are required whose miniaturization is technologically demanding. This makes such approaches suitable mainly for time and resource consuming laboratory based diagnostic. There is an increasing interest in Point of Care (PoC) analysis to perform diagnostic procedures, in a simple, low cost, rapid yet reliable fashion, at the site where it is needed: the doctor's office or even the patient's house. Direct electronic bio-recognition transduction involving a label-free process can offer a compelling alternative offering true miniaturization and easy data handling and processing. Low costs and versatility add up if organic electronic devices, such as organic field effect transistors (OFET) [2] are used as transducer. Recently OFET biosensors have shown, in fact, the potential to offer [3] very high performance level, while organic electronics allows to fabricate sensing circuits, also in an array configuration [4], on flexible, plastic or even paper, substrates by low cost printing compatible procedures. This can open interesting perspectives for the development of paper test-strip that combine low cost, reliability with label-free electronic detection and data processing. The sensing Field Effect Transistor approach (different from the ISFET one) has been so far actuated either through silicon nanowires or CNT nanoscopic devices or by using organic semiconductors in micro-meters size device. These approaches will be critically reviewed specifically considering their interfacing/integration with bio-system supramolecular architectures. Novel bio-FET will be presented where the immobilization of the biological recognition element is obtained by means of proteins incorporation into liposomes that are artificial lipid bilayers organized in vesicles which can provide nearly native environment for biomolecules. The spreading of small lipid vesicles on solid support allows in fact their self-assembling into fluid planar bilayers hosting the selected protein [5]. References

1. A.M. Bailey, D.J. Mitchell, K.L. Manjunath, G. Nolasco, C.L. Niblett FEMS Microbiology Letters 207 (2002) 153.

2. L. Torsi, A. Dodabalapur, Anal. Chem. 77 (2005) 380. 3. T. Someya, T. Sekitani, S. Iba, Y. Kato, H. Kawaguki, T. Sakurai, PNAS 101 (2004) 9966. 4. L. Torsi, et Al., Nature Materials 7 (2008) 412. 5. P. S. Cremer and S. G. Boxer, J. Phys. Chem. 103 (1999) 2554.

COLORIGRAMMI: IMPIEGO DI FOTOCAMERE DIGITALI COME S ENSORI OTTICI PER L’ANALISI DEL COLORE DI MATRICI ALIMENTARI DISOMOGE NEE

Alessandro Ulrici, Giorgia Foca

Dipartimento di Scienze Agrarie e degli Alimenti, Università di Modena e Reggio Emilia, Padiglione

Besta – Via Amendola, 2, 42100 Reggio Emilia (Italy) [email protected]

Spesso, durante l’acquisto di un prodotto alimentare, il consumatore è fortemente influenzato dal suo aspetto visivo, ovvero da una complessa combinazione di diverse caratteristiche quali il colore, la tessitura, l’eventuale presenza di difetti, la somiglianza con analoghi prodotti ritenuti “genuini” o con lo stesso tipo di prodotto “come è sempre stato”. Per queste ragioni, l’industria alimentare sta ponendo sempre maggiore attenzione al controllo ed all’ottimizzazione non solo del gusto e delle caratteristiche nutrizionali dei prodotti alimentari, ma anche del loro aspetto. A questo scopo è essenziale identificare metodi rapidi, oggettivi e riproducibili per quantificare caratteristiche visive dell’alimento, che possono a loro volta essere correlate con altre proprietà, come ad esempio la tipologia di materie prime impiegate, le quantità di ingredienti o le condizioni di processo. Inoltre, la definizione di relazioni quantitative tra aspetti legati al colore e parametri chimici (ad esempio, concentrazione di pigmenti o pH) può essere di aiuto nella comprensione della natura chimica della matrice analizzata e dei meccanismi responsabili delle modificazioni del suo aspetto, mentre l’individuazione di correlazioni con parametri sensoriali legati all’aspetto può portare alla messa a punto di un “occhio elettronico” veloce, oggettivo e trasferibile, che può essere di grande aiuto nel monitoraggio on-line. Ciò che rende ancora più interessante questa tipologia di approccio è il fatto che i sensori ottici necessari per raggiungere gli obiettivi illustrati sono già ampiamente disponibili a costi molto contenuti. Infatti, attualmente sul mercato è disponibile un vasto assortimento di fotocamere e videocamere digitali, webcam o scanner, capaci di fornire immagini RGB che permettono di convertire l’aspetto di un prodotto alimentare in una serie di dati. Il fattore chiave è quindi la definizione di appropriate metodiche per l’estrazione dell’informazione utile contenuta in questi dati ed il suo impiego in modelli di calibrazione, classificazione, monitoraggio di processo, ecc. In questo contesto, nel nostro gruppo di ricerca è stato sviluppato un algoritmo per l’estrazione e la quantificazione dell’informazione relativa al colore contenuta in immagini RGB, che essenzialmente consiste nel trasformare le immagini in altrettanti segnali monodimensionali chiamati colorigrammi [1], derivanti dall’unione in successione di curve di distribuzione di frequenza e di altri valori puntuali relativi a parametri legati al colore. I colorigrammi, che possono essere considerati alla stregua di fingerprint del colore delle immagini e sono quindi generati indipendentemente dalla natura specifica del prodotto analizzato e del problema in esame, possono essere analizzati con metodi chemiometrici di pattern recognition per l’esplorazione dei dati e per la creazione di modelli di calibrazione e classificazione. Questo approccio, già applicato a diverse tipologie di matrici alimentari (pesto alla genovese, prosciutti, uva, pane, omogeneizzati per l’infanzia, lasagne, ecc.) per lo studio di problematiche di varia natura (calibrazione di parametri analitici e sensoriali, classificazione di prodotti sulla base di diverse marche/cultivar/difetti, ecc.) ha generalmente portato a risultati soddisfacenti, anche a confronto con tecniche colorimetriche più tradizionali. Riferimenti 1. A. Antonelli, M. Cocchi, P. Fava, G. Foca, G.C. Franchini, D. Manzini, A. Ulrici, Anal. Chim. Acta 515

(2004) 3.

METAL OXIDE PARTICLES ON GLASSY CARBON AS NEW ELECT RODES FOR AMPEROMETRIC SENSORS

Fabio Terzi1, Josè M. Palacios-Santander2, Barbara Zanfrognini1, Laura Pigani1,

Chiara Zanardi1, Renato Seeber1

1 Department of Chemistry, University of Modena and Reggio Emilia, [email protected]

2Departamento de Química Analítica, Universidad de Cádiz, Apdo. 40, E-11510 Puerto Real (Cádiz)

Metal oxide particles are widely employed in catalysis. Unfortunately, most of them possess negligible electrical conductivity that prevents their use in electrocatalysis. In this work we show that metal oxide particles may enhance the electrocatalytic performance of an electrode surface, taking advantage of a bifunctional catalytic mechanism. Furthermore, grafting of these systems on the electrode surface is not straightforward and often constitutes the critical step in the use of these materials. An innovative method for anchoring commercial CeO2 and SnO2 particles to glassy carbon (GC) electrodes is presented, exploiting peculiar chemisorption phenomena of the metal oxides on the electrode surface. The analytical performances of the modified electrodes developed have been tested through different electrochemical techniques, in solutions of quite different benchmark analytes such as ascorbic acid (AA) and 2,4,6-tricholorophenol (TCF). AA has been chosen due to the importance of its determination in biological matrices. On the other hand, TCF is a very hazardous environmental pollutant whose determination by voltammetric techniques presents many problems, the electrode surface resulting fouled by polymerization of the electroactive species. The results obtained by the novel electrode material show i) increased sensitivity; ii) shift of the oxidation peaks to less positive potentials; iii) absence of fouling effects, in particular in the case of TCF, also thanks to suitable electrochemical cleaning procedure. Further improvements have been obtained through optimisation of the parameters of the pulsed electrochemical techniques employed for the analysis, namely Differential Pulse Voltammetry and SQuare Wave Voltammetry. Even better results are gained by suitable activation procedure of the GC surface: different activation methods were tested, both in acid and in basic media. The results show that the activation in sulphuric acid leads to best analytical performance. Finally, the morphology and composition of the different modified electrodes have been studied by means of electron microscopies coupled with Energy Dispersive Spectroscopy, X-Ray Diffraction and vibrational spectroscopies.

ABSTRACT

dei

POSTER

ENZYMATIC BIOSENSORS IN FOOD AND ENVIRONMENTAL FIEL DS

Marta Letizia Antonelli, Luigi Campanella, Dalina Lelo and R.F. Tornelli 1 Chemistry Department, SAPIENZA University Rome, Piazzale A. Moro, 5 – 00185 – Rome – Italy

[email protected]

Electrochemical biosensors in the last forty years have been widely studied [1]. More recently their analytical application in food and environment has been implemented. In particular, sensors based on enzymatic or cellular receptors have been employed for determining many different analytes such as: heavy metal ions, pollutants and pesticides, nutritional and/or safety markers as well as contaminants in food [1,2]. Some applications of electrochemical and calorimetric biosensors are here presented to determine heavy metals and chlorine containing pollutants for environmental purposes. Moreover the analysis of some sugars, hydroxyacids and lipids in foods by means of enzymatic biosensors is presented. Cellular-based amperometric biosensors have been developed some years ago to quantify some heavy metals, benzene and also chloro-phenol [3-5]. An ISE electrode has been designed and assembled basing on a specific membrane with immobilized lichens (Evernia prunastri) for Pb(II), Cu(II), Cr(VI) and 2-chloro-phenol determination. A similar amperometric sensor, based on immobilized Pseudomonas putida, was applied to benzene determination in urban area. A membrane electrode with immobilized yeasts (Saccharomyces cerevisiae) was applied to toxicity determination. More recently a new biosensor, working in a flow system and based on a Trehalase reactor combined with a GOD amperometric sensor, was applied to trehalose quantification in food [6]. Some microcalorimetric applications of enzyme-based biosensors were applied to test different foods. A microcalorimetric enzyme-based biosensor has been used for determining substances of interest in food, for the nutritional and safety aspects. In particular three hydroxyacids (Ascorbic, Orotic and Malic acid) have been determined in milk, sport drinks and vitamin tablets, by measuring the heat quantities associated with the enzymatic reactions involved, in the presence of the specific enzymes (Ascorbate oxidase, Orotate reductase and Fumarase) [7]. Also the triglycerides content in some foods (milk, oil and yoghurt) has been determined by the enzymatic decomposition of lipids in the presence of Lipase [8]. References 1. L.D. Mello and L.T. Kubota, Food Chem. (2002). 2. G. Hanrahan, D.G. Patil and J. Wang, J. Environ. Monit. 6 ( 2004) 657. 3. L. Campanella, G.Crescentini, M.G.D'onorio, G. Favero, M. Tomassetti, Annali di Chimica 86 (11-12)

(1996) 527. 4. M.L. Antonelli, P. Ercole and L. Campanella, Talanta 45 (1998) 1039. 5. M.L. Antonelli, P. Ercole and L. Campanella, Anal. Bioanal Chem. 381(5) (2005) 1041.

6. M.L. Antonelli, F. Arduini, A. Laganà, D. Moscone, V. Siliprandi, Biosens. Bioelectron. 24 (2009) 1382.

7. M.L. Antonelli and R.F. Tornelli, J. Therm. Anal. Cal. 91(1) (2008) 113.

8. L. Forte, G. Vinci and M.L. Antonelli, Anal. Lett. 29 (13) (1996) 2347.

TEMPLATE SYNTHESIS OF POLYANILINE NANOSTRUCTURES FOR SENSING APPLICATIONS

Francesca Berti 1, Silvia Todros 2, Giovanna Marrazza 1, Marco Mascini1, Guido Faglia2, Dhana

Lakshmi3, Iva Chianella3, Michael Whitcombe3, Sergey Piletsky 3, Antony P. F. Turner 3

1 Department of Chemistry, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Italy, [email protected] 2 CNR - INFM SENSOR Laboratory, Department of Chemistry and Physics,

University of Brescia, via Valotti 9, 25133 Brescia, Italy 3 Cranfield Health, Cranfield University,

Vincent Building, Cranfield, Bedfordshire, MK43 0AL, UK

The aim of this work was the development and characterisation of conductive polyaniline (PANI) nanostructures for sensing applications. The main challenge in developing electrochemical sensors based on nanostructures is the possibility to realise a reliable electrical contact and to optimise transduction from active sites, or receptors, to the electrode. Among conducting polymers, polyaniline (PANI) has generated great interest because of cheap and easy availability of raw materials for the synthesis, ease of processability, high conductivity and simple doping process. PANI nanostructures were obtained by electrochemical polymerisation of aniline monomers using alumina nanoporous membranes as template. A gold layer was sputtered on one side of the membrane employed as template in order to achieve electrical conductivity. After polymerization template was removed by dissolving the membrane in NaOH, thus obtaining a ready-to-use PANI-nanowires array sensor, with the gold layer as electrical contact. After an electrochemical and morphological (SEM) characterisation, nanostructures were grafted with receptors such as enzymes, antibodies, MIPs and used as highly sensitive affinity sensors.

SPECTROSCOPIC AND ELECTROCHEMICAL CHARACTERIZATION OF Ag NANOPARTICLES DISPERSED IN A POLYVINYL ALCOHOL FILM AND THEIR USE

FOR THE AMPEROMETRIC DETECTION OF GLUCOSE

Maria Rachele Guascito, Cosimino Malitesta, Daniela Chirizzi, Elisabetta Mazzotta, Rosaria Anna Picca

Laboratorio di Chimica Analitica, Dipartimento di Scienza dei Materiali, Università del Salento,

via Monteroni 73100 Lecce [email protected]

Nanoparticles have recently attracted a remarkable interest in the development of optical and electrochemical sensors, thanks to their dimensions that determine peculiar optical, electronic and catalytic properties. Chemically modified electrodes with nanoparticles of metals or oxides of metals have been proposed in order to improve sensitivity and limits of detection and also to fabricate miniaturized devices, as required in diagnostic applications [1-2]. Recently our research group proposed the application of Ag nanoparticles for the direct hydrogen peroxide detection [3]. In the present work, a new amperometric sensor based on a Pt electrode modified with Ag nanoparticles was developed. For the first time, Ag nanoparticles immobilization in a polyvinyl alcohol (PVA) film is proposed for the analytical determination of glucose in water. Up to now, few examples have been reported about the development of chemically modified electrodes based on metal nanoparticles [4] for the direct determination of glucose. The proposed nano-structured film was prepared by direct drop casting of synthesized Ag nanoparticles capped in a PVA colloidal suspension previously characterized by UV-vis spectroscopy. Glucose amperometric detection was performed at -0.4V in the concentration range 10 µM-30 mM. An extensive characterization of composite film was performed by X-ray Diffraction (XRD), Electronic Microscopy in Transmission (TEM), Photoelectron Spectroscopy (XPS) and Cyclic Voltammetry (CV). XRD and TEM analysis confirmed the presence of Ag nanodimensional particles dispersed in PVA matrix. XPS analysis was used to investigate both PVA film and nano-structured PVA film. C1s, O1s and Pt4f signals are evident on both films, while Ag3d signal is evident only on the nano-structured one, as expected. The binding energies of Ag3/2 and Ag5/2 electrons are 368.1 eV and 374.2 eV respectively, indicating that Ag nanoparticles in PVA film are in the Ag(0) state. CV experiments showed that Ag nanoparticles can give a pair of well-defined peaks with the formal potential of 0.25V vs SCE when the film is cycled in phosphate buffer (pH = 7) at scan rate of 25 mVs-1. Moreover, amperometric data evidenced that the presence of Ag nanoparticles is responsible for a remarkable increment in the current due to glucose oxidation. References 1. C. Welch, R.G. Compton, Anal. Bional. Chem. 384 (2006) 601. 2. X. Luo, A. Morrin, A.J. Killard, M. R. Smyth, Electroanalysis 18 (2006) 319. 3. M.R. Guascito, E. Filippo, C. Malitesta, D. Manno, A. Serra, A. Turco, Bios. Bioelectron. 24 (2008) 1057. 4. J. Lu, I. Do, L.T. Drzal, R.M. Worden, I. Lee, ACS Nano 2 (2008) 1825.

OPTIMIZATION OF METAL LAYERS NANOSTRUCTURE FOR DNA HYBRIDIZATION MONITORING IN A SPR IMAGING EXPERIMENT

Maria Grazia Manera1, Roberto Rella, Jolanda Spadavecchia, Julien Moreau, Michael Canva,

Andrey Savchenko

1 IMM-CNR Lecce, via per Monteroni 73100 Lecce, [email protected] 2Laboratoire Charles Fabry, Univ. Paris Sud, CNRS France,

Institute of Semiconductor Physics, National Academy of science, Kyiv, Ukreine

Biosensor approaches based on optical transducers are widely used in bioanalytical chemistry and in rapidly developing fields such as DNA chip and proteomics. Surface Plasmon Resonance (SPR) is a most attractive optical technique, which allows real-time monitoring of biochemical interactions without the need for labeling of reagents1. It is an optical technique for detecting changes in refractive index at the surface of a thin gold layer in contact with the biomolecules to be investigated. SPR can also be implemented into an imaging set-up (SPRI) to allow parallel characterization of an entire area, ranging typically from 10 mm2 to 1 cm2, for real-time multispot biochemical analyses, which can increase the throughput tremendously2. The choice of optimum parameters of the thin gold film are key problems for both optimal transformation of biochemical information into a signal and the formation of artificial interfacial architectures. The aim of this work is the study of the temperature influence on optical, structural and morphological properties of thin gold films as physical transducers of a SPRI biosensor. Gold films of 50 nm were deposited in vacuum by e-beam evaporation on onto SF10 slabs (25×25 mm2). After deposition, the samples were annealed at different temperatures (100, 150, 200, 250°C). Morphological and topographic features of the thin films annealed at different temperatures were analyzed using X-ray diffraction, Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). X-ray characterization shows a polycrystalline structure with a preferred orientation in the direction (1 1 1). With the increase of the annealing temperature the intensity of these reflection peaks increases. Moreover, morphological characterization revealed that the increase of the annealing temperature produces a pronounced packing density. DNA-DNA hybridization reaction was monitored using an home made Surface Plasmon Resonance Imaging setup (SPRI) based on a Krestchmann configuration (Figure 1). Biotinylated Dna probes, diluted at a concentration of 15µM in PBS were deposited using a microarrayer by using an immobilization procedure based on Dextran chemistry. A 20 bases

ssDNA sequence, which centre is perfectly matched to the common sequence found on the probes, is then injected in the cell at a concentration of 1 µM, in a PBS buffer. After rinsing, the mean hybridization response, averaged over all spots, obtained for different samples was recorded (Figure 1). A more than 2-fold variation in the hybridization signal is observed with annealing temperature, 200°C giving the highest response. Morphological investigations reveal that this annealing temperature gives compact films ideal for an efficient immobilization of biomolecules. The effect of a negative control and of regeneration of the biosensor was taken into account.

References 1. J. Homola, S. Yee, D. Myszka in F. S. Ligler, C.A.R. Taitt (Eds.), Surface Plasmon Resonance

Biosensors, Elsevier, The Netherlands, 2002, pp 207-252. 2. I. Mannelli et al., Biosensors and Bioelectronics 22 (2007) 803.

ELECTROCHEMICAL METHODS OF PNA/DNA HYBRIDIZATION ST UDIES

Ilaria Palchetti, Francesca Berti, Serena Laschi, Giovanna Marrazza, Marco Mascini

Dipartimento di Chimica, Università degli Studi di Firenze, Via della Lastruccia 3, 50019 Sesto Fiorentino (FI), Italy. [email protected]

Recently, there has been an increasing interest for DNA sensors based on the SAMs (Self Assembled Monolayers) of Peptide Nucleic Acid (PNA) modified electrodes. PNA has a neutral peptide-like backbone with nucleobases that allows the molecule to hybridize to complementary DNA strands with high affinity and specificity. In this communication, we present hybridization studies with DNA target oligonucleotides on a mixed monolayer of PNA and MCH (mercaptohexanol) on Au electrodes using EIS (Electrochemical Impedance Spectroscopy) and SECM (Scanning Electrochemical Microscopy). The immobilized PNA probes on the sensor surface are uncharged, and hence, do not affect the charge transfer from the redox mediator K4Fe(CN)6/K3Fe(CN)6 to the electrode. Once DNA targets hybridize to PNA, the charge density at the sensor surface will be changed. Thus, one can use EIS and SECM to conveniently monitor the PNA/DNA hybridization process in a label-free approach. Moreover electrochemical transduction of the hybridization process was also performed after coupling of a streptavidin–alkaline phosphatase conjugate and the bio-catalyzed precipitation of an insoluble and insulating product onto the surface of a gold electrode. As a consequence, the surface conductivity of the regions where hybridization had taken place decreased. This decrease can be adequately monitored by SECM and EIS.

MINIATURISED CARBON SENSOR FOR DIRECT ELECTROCHEMIC AL

DETERMINATION OF HYDROXYMETHYLFURFURAL (HMF)

M. Mascini1, M. Del Carlo1, A. Amine3, A. Piva1, G Dimitri2 and D. Compagnone1

1Department of Food Science, University of Teramo, 64023 Teramo, Italy [email protected]

2 Consorzio Istituto Nazionale Biostrutture e Biosistemi (I.N.B.B.),00136 Roma ,Italy 3 Université Hassan II – Mohammedia, B.P. 146, Mohammedia, Morocco

HMF content is used by many countries as a food quality indicator, for example honey with more than 40 mg/L HMF is rejected [1]. Analytical techniques to quantify HMF content are manly based on indirect analysis method of derivatives requiring special treatment of samples using spectrophotometry (visible or ultraviolet) and high performance liquid chromatography (HPLC). As alternative to traditional spectrochemical techniques for the detection of HMF in routine analysis we propose the use of electroanalytical techniques coupled with disposable screen-printed electrodes [2]. There are few electrochemical studies of HMF, one of the most recent reporting a direct determination of HMF using a mercury drop electrode by means of electrochemical reduction of HMF [3]. However because of its toxicity the use of mercury has been banned by the European community. Following the approach proposed by Reyes-Salas et al. [3] a new miniaturized electrochemical carbon sensor for HMF analysis was optimized, avoiding the use of mercury as working electrode. Coupling this sensor with differential pulse cathodic voltammetry (DPV) a detection limit of 0.8 mg/L with a sensibility of 800 nA/(mg/L) for HMF were found. Deoxygenation of sample solution was not necessary. The entire analysis time was shorter than 2 min. No special sample treatment was required, just a dilution in borate buffer (0.5M, pH 9.1) used as supporting electrolyte because it allowed better definition and selectivity of the HMF reduction signal at around -1400mV vs. Ag/AgCl. Examples of food applications like honey and cooked must was carried out. The detection was performed using standard addition method. References

1. S. Bogdanov, Bee World 80 (1999) 61. 2. I. Palchetti, S. Laschi, M. Mascini, Analytica Chimica Acta 530 (2005) 61. 3. E. Reyes-Salas, J. Manzanilla-Cano, M. Barceló-Quintal, D. Juárez-Mendoza, M. Reyes-Salas,

Analytical Letters 39 (2006) 161. This work was supported by the 7th FRAMEWORK PROGRAMME Marie Curie Actions People IRSES N°230815 NANOSENS and by Consorzio Interuniversitar io INBB research fellowship 2008.

ENHANCEMENT OF THE IMPRINTING EFFECT OF A Co-PORPHY RIN BASED MOLECULARLY IMPRINTED POLYMER

BY THE OPTIMIZATION OF WASHING PROCEDURE

Elisabetta Mazzotta, Cosimino Malitesta

Laboratorio di Chimica Analitica, Dipartimento di Scienza dei Materiali, Università del Salento, via Monteroni 73100 Lecce [email protected]

One of the most important issues of imprinting technology is the development of materials having a high degree of selectivity and presenting fast desorption and rebinding kinetics under mild conditions [1]. The most common imprinting approaches are based on covalent and noncovalent interactions, each of one offering different levels of specificity and reversibility. Recently, research has been devoted to the development of alternative imprinting schemes based on new monomer-template interactions. Among these, metal-coordination interaction offers promising opportunities to design novel MIPs. It combines in fact the flexibility of noncovalent approach with the strength and specificity of covalent one [2]. Moreover, the availability of a vast array of ligands allows to control and define the coordinating properties of metal centres towards different substrates [1]. Wide applications of metal complexes based MIPs in fields ranging from sensors [3] and catalysts [4] to enantioselective separations [5] are now reported. In all cases these materials have been prepared by chemical synthesis, while electropolymerization has never been used before for the synthesis of MIPs having a metal complex as recognition center. We have recently proposed the first attempt to develop an electrosynthesized MIP based on a metal complex with the aim to combine advantages coming from metal-ion coordination with the ones deriving from MIP electropolymerization [6]. A Co-porphyrin (Co(III)tetrakis(o-aminophenyl) porphyrin (Co(TAPP)) has been used as monomer and 2-(2,4-dichlorophenoxy) butyric acid (2,4-DB) [7] as template, producing an electrochemical sensor for 2,4-DB. In the present work efforts devoted to the optimization of the imprinting protocol will be described. As it has been observed that the electrochemical response is greatly influenced by the different extent of template removal, various washing procedures have been tested and compared in order to select the one producing the highest template removal without compromising MIP ability to selectively interact with 2,4-DB. The effect of different solvents as well as of the exposure time to washing mixture has been evaluated. XPS characterization of pristine and washed MIPs allowed to quantitatively estimate the amount of the withdrawn template and to analyze modifications occurring in MIP structure after washing step that could potentially influence its rebinding properties. References 1. P.K. Dhal, 2001 In: Sellergren B. (Ed.), Man-made mimics of antibodies and their applications in

analytical chemistry, Molecularly Imprinted Polymers. Elsevier Science, Amsterdam, pp.185–201. 2. A. Ersoz, A. Denizli, A. Ozcan, R. Say, Biosens. Bioelectron. 20 (2005) 2197. 3. B.R. Hart, K.J. Shea, J. Am. Chem. Soc. 123 (2001) 2072. 4. K. Sode, S. Ohta, Y. Yanai, T. Yamazaki, Biosens. Bioelectron. 18 (2003) 1485. 5. A.A. Oezcan, R. Say, A. Denizli, A. Ersoez, Anal. Chem. 78 (2006) 7253. 6. C. Malitesta, I. Losito, P.G. Zambonin, Anal. Chem. 71 (1999) 1366. 7. E. Mazzotta, C. Malitesta, Sens. Lett. 6 (2008) 618.

DEVELOPMENT OF A MINIATURIZED MULTIPLEXED ANALYTICA L DEVICE EMPLOYING LUMINESCENCE “CONTACT” IMAGING DETECTION

Aldo Roda1, Mara Mirasoli1, Luisa Stella Dolci1, Angela Buragina1, Patrizia Simoni2

1Department of Pharmaceutical Sciences, University of Bologna, via Belmeloro 6, 40126 Bologna [email protected]

2Department of Clinical Medicine, University of Bologna, via Massarenti 9, 40138 Bologna The accurate diagnosis of a given pathology requires the simultaneous detection of several biomarkers, thus prompting the development of “panel tests”. For this purpose, a great effort is devoted to the development of miniaturized devices able to perform accurate quantitative detection of several analytes in one sample, with high sensitivity and short analysis time. To reach this goal, the combination of biospecific analyte recognition reactions (e.g. immunological reactions, nucleic acids hybridization, enzyme reactions) with highly sensitive luminescence detection (bio-, chemiluminescence, and electrogenerated chemiluminescence) promises to be a successful strategy [1]. By using this approach, we propose the development of a miniaturized integrated analytical device, where the analytes are captured by biospecific probes arrayed on a transparent support and revealed by means of a second set of biospecific probes labelled with suitable luminescent tracers. With this “position encoding” approach, spatial localization of the analytical signal provides analytes identification, while its intensity is employed for their quantification through the use of internal standards. To obtain high assay sensitivity, luminescence “contact” imaging detection was employed, by placing the support in contact with a cooled CCD or with a CMOS device, able to localize and quantify the luminescent signal. The use of a fiber-optic taper as optical coupling element increased the resolution of contact detection, and consequently multiplexing possibilities. For this purpose, bio- and chemiluminescent labels with improved characteristics suitable for miniaturized analytical devices and multiplexed assays were developed: bioluminescence labels characterized by thermostability and red-shifted emission spectra were obtained by cloning new luciferase genes and by producing new luciferase mutants [2], while the enhancer/luminol/oxidant chemiluminescent cocktail for horseradish peroxidase was optimized to increase assays sensitivity [3]. Furthermore, we recently designed a transparent electrochemical cell suitable to perform imaging detection with electrogenerated chemiluminescence detection [4]. To increase multiplexing possibilities, the “position encoding” approach can be combined with a “signal encoding” approach, where different luminescent labels that can be independently measured (e.g. by sequential trigger of the luminescence reaction or by spectrally resolving signal emission) are associated to different analytes captured in the same area. To obtain a fully integrated and functional device, parallel developments in the design of a miniaturized module for on-line sample pre-analytical treatment and enrichment is the subject of other activities of the group. References 1. A. Roda, M. Guardigli, M. Mirasoli, E. Michelini, L.S. Dolci, M. Musiani, Luminescence 23 (2008) 89 2. E. Michelini, L. Cevenini, L. Mezzanotte, D. Ablamsky, T. Southworth, B. Branchini, A. Roda, Analytical

Chemistry 80 (2008) 260 3. E. Marzocchi, S. Grilli, L. Della Ciana, L. Prodi, M. Mirasoli, A. Roda, Analytical Biochemistry 377 (2008)

189 4. L.S. Dolci, S. Zanarini, L. Della Ciana, F. Paolucci, A. Roda. Analytical Chemistry (2009) DOI:

10.1021/ac900756a

DEVELOPMENT OF A PHOTOACOUSTIC SENSOR EMPLOYING SIL ICON BASED MICROCOMPONENTS FOR MEASURING 13CO2/12CO2 RATIO IN BREATH

Luca Belsito1, Mara Mirasoli2, Alberto Roncaglia1, Aldo Roda2

1IMM Bologna (CNR), Via Gobetti 101, 40129 Bologna

2Department of Pharmaceutical Sciences, University of Bologna, via Belmeloro 6, 40126 Bologna, [email protected]

The 13C-breath tests are non-invasive diagnostic techniques with various applications in gastroenterology, such as the diagnosis of Helicobacter pylori infection, the measurement of the gastric emptying time and of the oro-caecal transit time, the evaluation of hepatic or pancreatic functionality. The test is based on the measurement of the 13C/12C isotope ratio in expired CO2 at different times after administration to the patient of a suitably 13C-labelled substrate. This substrate, once metabolized in the target organ, leads to the formation of 13CO2, which is released in breath. To perform measurement of the 13CO2/

12CO2 ratio in breath, currently available instrumentation is based either on mass spectrometry or on non-dispersive infrared spectroscopy (NDIR). In particular, NDIR instruments, which exploit the IR absorbance of 12CO2 and 13CO2 in the range 2-8 µm, employ an IR source coupled with a chopper to alternate the analysis over two channels, each equipped with a photoacustic detection containing either 12CO2 or 13CO2. The use of miniaturized components and the elimination of mechanical parts in motion (chopper) should allow the construction of a portable instrument that can be more flexibly applied on the field. For this purpose, a preliminary feasibility study has been carried out aimed at realizing a photoacoustic gas sensing system based on microcomponents, starting from the working specifications of MEMS (Micro Electro Mechanical System) infrared emitters and pressure micro-sensors developed at the IMM Institute (CNR) in Bologna. In order to calculate the expected performances, a simulation model has been developed to estimate the pressure generated in a photoacoustic cell containing the gas of interest at a known pressure [1], considering both the case of 13CO2 and 12CO2. In this model, the calculation of the radiation absorption per unit length of optical path has been implemented using the software Hitran [2], taking into account all the absorption lines of the target gases in the 3-5 µm atmospheric window. Moreover, the sound pressure attenuation within the cell due to the presence of a known concentration of target gas in a 20 cm long optical path has been evaluated with the model in order to determine the expected signal to noise ratio. The results of the simulations have shown a strong dependence of the signal to noise ratio by the gas pressure inside the photoacoustic cell, due to which it was verified that a minimum chamber working pressure around 10 Torr is necessary to achieve a detection limit of 1 ppm for 13CO2. To operate in such conditions, it will be necessary to optimize the performance of the micro pressure sensor with a new design in order to increase their sensitivity in the optimal working point. The performance of the available micro-emitters, instead, is suited to the purpose according to the model results. References 1. L.B. Kreuzer, Journal of Applied Physics 42 (1971) 2937. 2. HITRAN data base [ http://www.cfa.harvard.edu/hitran/]

FURTHER APPLICATIONS OF A TYROSINASE-LACCASE BIOSEN SOR FOR PHENOLIC COMPOUNDS: ANALYSIS OF RIVER AND WELL WATE RS

Maria Rita Montereali, Livia Della Seta, Walter Vastarella, Carlo Cremisini

ENEA ACS PROTCHIM - CR Casaccia, Via Anguillarese 301- 00123 S.Maria di Galeria - Roma

(Italy), [email protected]

Phenol and substituted phenolic compounds are dangerous pollutants widely distributed in the environment. Most of the phenolic compounds are used or synthesized in several industrial processes, in petroleum refineries and in bleaching processes of paper industry. Halogenated phenolic compounds often arise from the chlorination of waters and (especially chlorophenols) can affect the taste and odour of drinking water at concentrations down to 1 µgL-1. Chlorinated phenolic derivates have also been found as main hydrolysis and photolysis products of chlorinated phenoxy acid herbicides (1). On the other hand, phenols can be produced from the decomposition of natural occurring substances like humic acids, lignin and tannins (2). Since phenol and phenolic derivates are extremely persistent in different environmental media and potentially toxic to human health and aquatic life in natural waters, it is particularly interesting to detect their content in various water samples. The official analytical methods used for the determination and identification of phenolic compounds in waters are based on liquid-liquid extraction followed by liquid or gas chromatographic detection (3). These methods have the major disadvantage of needing hazardous organic solvents, complex and time-consuming sample pre-treatment procedures and are unsuitable for on site analyses. The total content of phenols in natural waters and wastewaters is also obtained by using the 4 aminoantipyrine (4AAP) colorimetric standard procedure (4). Biosensors, on the other hand, represent useful devices to carry out measurements directly on field, allowing a preliminary monitoring of water samples in order to identify the “positive” ones, that can be submitted successively to a further chromatographic detection. We propose a cheap, disposable amperometric biosensor based on the two enzymes Tyrosinase and Laccase (5, 6, 7), entrapped in a sol gel of diglycerylsilane and immobilized on graphite screen printed electrodes modified with ferrocene (8), for the detection of phenolic compounds in river and ground waters. Such a biosensor is easy to use and short time consuming and, although it is not able to distinguish single phenolic compounds and detect the orto-substituted, because of its enzymatic mechanism, its analytical results represent a fair estimate of total phenolic content. It has already been employed in the analysis of must and wine samples with good results and it can be used as an useful index and a versatile tool for total phenolic content estimate in both food and environmental matrices. The measurements have been performed using a portable lab-developed potentiostat (Domotek) and placing the sensor in a perspex homemade flow cell. The best operational values for applied potential, working pH and cell flow rate have been studied in order to establish the most effective conditions for the analysis of real samples and operational stability, reproducibility and repeatability have been tested. Sensitivity values, limits of detection and range of linearity of the biosensor measuring phenol and some phenolic compounds (chloro-, dihydroxy- and alkyl- phenolic derivates listed as priority pollutants) have been calculated and, to account for the possibility of matrix effect, recovery tests in real samples (river and well water) have been carried out using the standard addition method. References 1. Leo M. L. Nollet, Handbook of water analysis, by CRC Press (2000) 347. 2. Samuel D. Faust, Osman M. Aly, Chemistry of water treatment 2nd ed. by CRC Press (1998) 76. 3. D. Puig, D Barcelo, Trends in analytical chemistry 15 (1996) 362. 4. ASTM: Annual Book of ASTM Standards, Part 31, Water, Method D (1981) 1783. 5. J. C. Garcia-Borron, F. Solano, Pigment Cell. Res. 15 (2002) 162. 6. N. Durán, M. A. Rosa, A. D’Annibale, L. Gianfreda, Enzyme and Microbial Technology 31 (2002) 907. 7. C. F. Thurston, Microbiology 140 (1994) 19. 8. M. R. Montereali, W. Vastarella, L. Della Seta, R. Pilloton, Intern. J. Environ. Anal. Chem. 85 (2005) 795.

NANOWIRES IBRIDI DI Ni/Au PER CONCENTRARE ED ORIENT ARE BIOMOLECOLE SULLA SUPERFICIE DI TRASDUTTORI DI SEGNALE

Federica Valentini, Giuseppe Palleschi, Danila Moscone

Dipartimento di Scienze e Tecnologie Chimiche, Università di Roma Tor Vergata,Via della Ricerca

Scientifica, 00133 Roma; [email protected]

In molti campi della tecnologia la miniaturizzazione è diventata una necessità sempre più stringente, al fine di avere sistemi sempre più piccoli ed allo stesso tempo sempre più potenti. Di grande interesse sono le strutture aventi le dimensioni da 1 a 100 nanometri, per le loro particolari proprietà. L’elettrodeposizione è un metodo poco costoso, conveniente, e versatile per la fabbricazione di una vasta gamma di film metallici, semiconduttori, polimerici, e multistrato. Effettuando una elettrodeposizione multistrato in una membrana porosa di ossido di alluminio anodizzato che agisce da template, possono essere prodotti nanowires ibridi con dimensioni specifiche e controllate. Questo processo è definito infatti una “template synthesis” poiché prevede la modifica via sputtering della membrana di allumina con uno strato sottile di Ag (20-30 nm), che funzionerà dunque da contatto elettrico per l’elettrodeposizione. All’interno dei pori di questa membrana crescono i nanowires. Successivamente, tale membrana è rimossa mediante trattamento in soluzione alcalina concentrata. I nanowires invece, non solubili in questo medium, vengono raccolti in soluzione acquosa sottoforma di dispersioni stabili. In particolare, in questo lavoro sono stati sintetizzati per via elettrochimica (tramite cronoamperometria), nanowires intermetallici formati da segmenti di nickel e oro alternati, aventi una morfologia finale controllata dalla quantità di carica richiesta per la deposizione, dal potenziale di lavoro, dal tempo di sintesi e dalla tipologia di membrana usata come stampo per la crescita1. La presenza del nano-segmento di nichel offre un vantaggio notevole legato alle intrinseche proprietà magnetiche, utili per molte applicazioni analitiche, tra cui la possibilità di concentrare bioelementi (es. anticorpi, enzimi, DNA e RNA) sulla superficie di un elettrodo, (ma anche per veicolare farmaci, geni umani e virus all’interno di cellule), mediante l’applicazione di un campo magnetico esterno. Per immobilizzare tali biocomponenti, è necessario realizzare una geometria ibrida, che preveda la presenza di un nano-segmento di oro accoppiato all’elemento paramagnetico. L’oro, come è noto, risulta essere caratterizzato da un’elevata affinità nei confronti di svariati elementi biologici (esso è infatti ampiamente utilizzato per assemblare biosensori e per lo sviluppo di biosaggi). I nanowires ottenuti modificando diversi parametri della elettrosintesi, saranno caratterizzati morfologicamente attraverso diverse tecniche microscopiche quali SEM, TEM, AFM, ed attraverso tecniche spettroscopiche quali XRD, FTIR, e RAMAN per la caratterizzazione strutturale. In seguito, tali nanowires saranno utilizzati per assemblare immunosensori per la determinazione della tossina botulinica e sensori a DNA per la determinazione dell’Hepatovirus. Riferimenti

1. J. Wang, M. Scampicchio, R. Laocharoensuk, F. Valentini, O. González-García, J. Burdick, Journal of American Chemical Society 128 (2006) 4562.

Questo lavoro è svolto nell’ambito del progetto PRIN 07, prot. 2007AWK85F

FIELD-FLOW FRACTIONATION ASSISTED OLFACTORY SENSOR FOR PATHOGENIC BACTERIA IDENTIFICATION

Paolo Nanni1, Barbara Roda2, Pierluigi Reschiglian2, Aldo Roda1

1Department of Pharmaceutical Sciences, Via Belmeloro, 6, I-40126 Bologna, Italy,

[email protected] 2Department of Chemistry “G. Ciamician”, Via Selmi, 2, I-40126 Bologna, Italy

Each year, as many as 80 million cases of foodborne illness occur in United States and 1.5 millions in Italy, among these 30% are caused by bacteria and their related toxic products. Conventional microbiological methods for the identification of pathogenic bacteria are labor-intensive (several enrichment steps) and time-consuming (two to three days to obtain results). Therefore simpler, faster, more sensitive diagnostic techniques able to give on-site bacterial monitoring are required in order to improve the food safety (food production, processing, storage and distribution) as well as to screen for potential bacteria infection in humans. Recent efforts in the development of rapid detection systems are primarily focused on enzyme linked immunosorbent assay (ELISA) and PCR-based methods. Despite the significant reduction in assay time and improvement in detection limit of these rapid methods, there are still key issues that limit their widespread implementation. Among these issues are cost, simplicity, throughput and the inability to differentiate between live and dead cells (since dead cells are usually not pathogenic). Field-flow fractionation techniques (FFF) are flow-assisted separative techniques suitable for the fractionation of cells based on their morphological features [1]. FFF have been already successfully applied to the fractionation of different bacterial species, also before specific analysis with mass spectrometry. Moreover, FFF have already demonstrated its ability to differently sort viable and not viable bacteria. This feature, with the soft separation mechanism which permits to maintain inalterate viability and cell properties during fractionation process, make FFF techniques as useful tool for sample preparation for more specific analysis with a significant increase of analytical output. The simplicity of separation device allows for its implementation in portable and compact analytical systems for on-site identification. In this work the use of FFF system coupled to an electronic olfactory system (EOS) for the olfactive analysis of pathogenic bacteria is presented [2]. The EOS is equipped with six metal oxide semiconductor sensors and allows the qualitative analysis of the volatile products patterns of bacteria, which can be afterwards analyzed by chemometric analysis using Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA) for the identification and discrimination of bacteria species. To set up the method two bacterial species most frequently responsible for food-related illnesses were considered: E. coli O157:H7 (found in meat) and Yersinia enterocolitica (found in meat, milk). Bacteria cells mixtures with different bacteria proportions (1:4; 1:1; 4:1) were suspended in phyisiological buffer and directly injected in an FFF system at a final concentration of 108 CFU/ml. Fractions corresponding to the retention time typical for the two bacteria species were collected. Standard suspensions of Y. enterocolitica and E.coli and fractions collected were grown in Luria Bertani (LB) agar media overnight at 37°C and then analyzed with the olfactive system. The data recorded for each sample were subjected to chemometrical analysis using PARVUS software [3] and the selection of the variable relevant for the discrimination among the classes of bacteria was performed using SELECT. The selection of 10 variables allowed to clearly discriminate by PCA the Y. enterocolitica and E.coli samples, and the LDA analysis allowed to obtain a correct classification and prediction ability of respectively 100 and 87.5 %. The analysis of collected fractions from the different mixtures confirms that after fractionation, the olfactory system was able to distinguish and identify the different fractions. The development of an on-line FFF coupling with the olfactory system for the rapid and simple identification of pathogenic bacteria is in progress. References 1. P. Reschiglian, A. Zattoni, B. Roda, E. Michelini, A. Roda, Trends in biotechnology 23 (2005) 475. 2. P. Pasini, N. Powar, R. Gutierrez-Osuna, S. Daunert, A. Roda, Anal Bioanal Chem. 378(1) (2004) 76. 3. M. Forina, S. Lanteri, C. Armanino, M.C.C. Oliveros, C. Casolino. V-PARVUS 2004. Freely available at

http://www.parvs.unige.it

VALIDAZIONE DI UN METODO BASATO SULL’ELETTROCHEMILU MINESCENZA PER DETERMINARE GLI E° DI AMMINE ALIFATICHE TERZIAR IE

CON L’USO DELLA TEORIA DI MARCUS

Denis Badocco1, Stefano Pegoraro1, Paolo Pastore1

1 Dipartimento di Scienze Chimiche, Università di Padova, Via Marzolo n°1, 35125 Padova, [email protected]

Tramite l’utilizzo della tecnica chiamata “flash Elettrochemiluminescenza (ECL)”1 è stato

possibile determinare in maniera semplice il potenziale redox di un insieme di ammine alifatiche terziarie (R3N). La “Flash ECL” consiste nell’applicazione di un impulso di potenziale molto breve (50µs) ad un microelettrodo (raggio 4µm) immerso in una soluzione tamponata contenente un sistema ECL quale Ru(dpp)3

2+/R3N. La forma dei profili di emissione ottenuti contiene importanti informazioni sulla natura cinetica dello stadio lento del meccanismo ECL e sulla termodinamica delle reazioni coinvolte. In particolare l’area di emissione è proporzionale al potenziale redox delle ammine utilizzate. E’ stato così possibile ottenere il potenziale redox delle seguenti ammine: trietilammina, tributilammina, triisobutilammina, trimetilammina, metildipropilammina usando come riferimento l’E° della tripropilammina. In questo c ontesto ci riproponiamo di convalidare l’uso di questa tecnica ricavando l’E° delle ammine attraver so l’uso di una metodologia basata sull’applicazione della teoria di Marcus2. L’approccio sperimentale è lungo e le misure elettrochimiche sono condizionate dalla scelta dei mediatori redox e dalla limitata solubilità delle ammine alifatiche in soluzione acquosa. In questo contesto verranno definite delle regioni di lavoro ottimali per la valutazione delle cinetiche in fase omogenea. Tali regioni sono definite da parametri sperimentali come il pH, la concentrazione di mediatore e di ammina alifatica. Sperimentalmente, dalla misura di correnti limite dell’ossidazione catalitica di diversi mediatori redox quali Ru(CN)6

4-, Fe(bpy)32+, Ru(bpy)3

2+, Ru(phen)32+ al variare della concentrazione di R3N,

su un microelettrodo di Pt, si ottiene la costante di velocità del trasferimento elettronico omogeneo (km). La figura 1 mostra una curva ottenuta con l’equazione di Marcus fittando i dati sperimentali di log km verso E°R/Q – wp (potenziale di ossidazione dei mediatori redox a cui viene sottratto il termine elettrostatico) ottenuti con i quattro mediatori e la tripropilammina. Il fitting ha permesso di ottenere un E° di 0.91V vs SCE.

1.0 1.1 1.2 1.34

5

6

7

8

log

k m

E°R/Q

- wp (V vs NHE)

Figura 1. Plot di Marcus (linea continua) i cerchi vuoti rappresentano i valori di log km ottenuti con i quattro mediatori seguenti: Ru(CN)6

4-, Fe(bpy)32+, Ru(bpy)3

2+ e Ru(phen)32+. Le curve tratteggiate rappresentano

le bande di confidenza al 95% (più strette) e le bande di predizione, sempre al 95% (più larghe). Riferimenti 1. D. Badocco, F. Zanon, P. Pastore, Electrochimica Acta 51 (2006) 6442. 2. F. Kanoufi, Y. Zu, A. J. Bard, J. Phys. Chem. B 105 (2001) 210.

ELECTROSYNTHESISED POLYIMIDAZOLES FOR SENSORS BASED ON MOLECULAR IMPRINTING

Rosaria Anna Picca, Cosimino Malitesta

Laboratorio di Chimica Analitica, Dipartimento di Scienza dei Materiali, Università del Salento, Via

Monteroni, 73100 Lecce (Italy) [email protected]

The electropolymerisation of imidazole (and its derivatives) onto Pt electrodes was proposed in [1, 2] but, unlike the synthesis of polypyrroles or polythiophenes, it has not been extensively exploited to prepare chemical sensors. In fact in this field polyimidazoles have been prepared by classical methods and have shown also good electroactivity working as “electrochemical wires” in enzyme biosensors [3] as well as great affinity as ligands towards some metals such as Cu2+ [4]. Moreover the coupling of electrochemical polymerisation and Molecular Imprinting (MI) for the preparation of selective materials towards target molecules have demonstrated particularly appealing to assemble polymeric films, acting as sensing layers, directly onto the surface of a transducer (for reviews see [5, 6]). Also regarding imidazoles in MI, 1-vinylimidazole (especially in complexes with transition metals) represents still the most successfully used [7, 8] in classical synthetic schemes. Based on our previous work on electrosynthesised molecularly imprinted polymers [9] in this communication we present preliminary attempts to couple the electrochemical synthesis of imidazole (as model monomer) with imprinting of small molecules (e.g. glucose, catechol, …) onto electrodes and their possible application to develop novel sensors for such targets. References 1. H.-L. Wang, R.M. O'Malley, J.E. Fernandez Macromolecules 27 (1994) 893. 2. L. Martinot, l.D. Léroy, C. Jerôme, O. Leruth Journal of Radioanalytical and Nuclear Chemistry 224

(1997) 71. 3. T.J. Ohara, R. Rajagopalan, A. Heller, Analytical Chemistry 65 (1993) 3512. 4. T. Yamamoto, T. Uemura, A. Tanimoto, S. Sasaki, Macromolecules 36 (2003) 1047. 5. M.C. Blanco-López, M.J. Lobo-Castañón, A.J. Miranda-Ordieres, P. Tuñón-Blanco, Trends in Anaytical

Chemistry 23 (2004) 36. 6. A. Merkoçi, S. Alegret, TrAC- Trends in Anaytical Chemistry 21 (2002) 717. 7. S. Vidyasankar, F.H. Arnold, Current Opinion in Biotechnology 6 (1995) 218 (and refs. therein). 8. T. Yamazaki, E. Yilmaz, K.Mosbach, K. Sode, Analytica Chimica Acta 435 (2001) 209. 9. C. Malitesta, I. Losito, P.G. Zambonin, Analytical Chemistry 71 (1999) 1366.

A D-AMINO ACID OXIDASE MICROBIOSENSOR FOR D-SERINE DETECTION IN CENTRAL NERVOUS SYSTEM

Loredano POLLEGIONI1, Mirella PILONE1, Pierre PERNOT2,3, Oleg SHUVAILO3,4, Alexey SOLDATKIN5, Jean-Pierre MOTHET6, Raymond CESPUGLIO3, Stéphane MARINESCO3

1The Protein Factory, Dept. Biotechnology Molecular Biology, University of Insubria, Varese, Italy

[email protected] 2CNRS UPR 9040, 1 avenue de la Terrasse, Gif-sur-Yvette, France

3INSERM U628, Université Claude Bernard Lyon I, Lyon, France 4EA3734, Université Claude Bernard Lyon I, 8 avenue Rockefeller, 69373 Lyon, France

5Institute Molecular Biology & Genetics, National Academy of Science of Ukraine, Kiev, Ukraine 6Institut Francois Magendie, INSERM U378, 33077 Bordeaux

Large amounts of D-serine are involved in neurotransmission in the frontal brain areas. D-serine is released by astrocytes in response to chemical stimulation by glutamate, or glutamate receptor agonists. D-serine is an endogenous agonist of the glycine site of the N-methyl-D-aspartate (NMDA) receptors, and plays important roles in synaptic plasticity and in several neurological disorders, such as schizophrenia, amyotrophic lateral sclerosis, ischemia, and Alzheimer’s disease. It is therefore an important target for the pharmaceutical industry [1,2]. Notwithstanding the physiological importance of D-serine under normal and pathological conditions, the variations in D-serine levels in brain during behavior or pathological states remain largely unexplored. Up to now, detection of free D-serine in the central nervous system has been performed using microdialysis which is limited by the large size of the probes used to collect brain samples and by the slow diffusion of molecules through the dialysis membrane. To overcome these technical limitations, we developed an enzymatic microbiosensor capable of detecting D-serine in vivo [3]. This technique relies on cylindrical platinum microelectrodes (150×25 µm), covered with a membrane of poly-phenylenediamine (PPD), and a layer of D-amino acid oxidase from the yeast R. gracilis (RgDAAO). This enzyme is a FAD-containing flavoprotein that catalyzes the oxidative deamination of D-isomers of neutral, and with lower efficiency, basic D-amino acids. It is characterized by a high specific activity, tight binding with the cofactor, high stability in the immobilized form and high production levels as recombinant protein [4]. RgDAAO was immobilized by cross-linking with bovine serum albumin using glutaraldehyde: it is used to convert D-serine into hydroxypyruvate and ammonia, while producing hydrogen peroxide that, in turn, is oxidized by the electrode. The microbiosensor measures D-serine levels with a 100 nM detection limit under +500 mV constant potential vs. Ag/AgCl. For an injection of 1 µM D-serine, the microbiosensor produces an increase in current response, reaching 90 % of its steady state current within about 2 s. PPD provides a more selective membrane than other phenylenediamine isomers. Interferences by biogenic amines and their metabolites, ascorbic acid, uric acid and L-cysteine are greatly reduced by the PPD layer. Furthermore, this biosensor is highly specific since D-serine is the only RgDAAO substrate present in significant amounts in the central nervous system. D-serine levels estimated by the microbiosensor in brain extracts were similar (+6%) to the values measured by liquid chromatography. In conclusion, this method will make it possible to study in vivo the function(s) of D-serine, as well as to assess the pharmacological potency of new drugs designed to impact D-serine metabolism in neurological or psychiatric disorders such as schizophrenia [5]. References 1. S.O. Oliet, J.P. Mothet, Glia 54 (2006) 726. 2. A.K. Mustafa, P.M. Kim, S.H. Snyder, Neuron Glia Biol 1 (2004) 275. 3. P. Pernot, JP. Mothet, O. Schuvailo, A. Soldatkin, L. Pollegioni, M. Pilone, MT. Adeline, R. Cespuglio, S.

Marinesco, Anal Chem. 80 (2008) 1589. 4. L. Pollegioni, S. Sacchi, L. Caldinelli, A. Boselli, MS. Pilone, L. Piubelli, G. Molla, Curr Protein Pept Sci. 8

(2007) 600. 5. S. Sacchi, MG. Bernasconi, M. Martineau, JP. Mothet, M. Ruzzene, MS. Pilone, L. Pollegioni, G. Molla, J

Biol Chem. 283 (2008) 22244.

ELETTRODO CHIMICAMENTE MODIFICATO CON Co-A. CARATTERIZZAZIONE E APPLICAZIONI ANALITICHE.

Francesca Zavarise, Daniele Merli, Maria Pesavento, Antonella Profumo

1 Dipartimento di Chimica Generale, Università degli Studi di Pavia, via Taramelli 12, 27100, Pavia,

[email protected]

Molecole biologiche quali amminoacidi, peptidi o enzimi sono in grado di interagire con ioni metallici. In letteratura sono riportati molti studi su elettrodi chimicamente modificati (CME) ottenuti con substrati biologici che possono essere utilizzati per determinazioni analitiche [1-3]. Tra questi il coenzima A [CoA] ha buone proprietà complessanti nei confronti dei metalli pesanti per la presenza nella molecola di gruppi fosfato e del gruppo adenosinico. Allo stesso tempo può essere facilmente utilizzato per preparare CME sfruttando la presenza di un gruppo tiolico che può legarsi covalentemente con la superficie d’oro di un elettrodo solido secondo la seguente reazione:

RSH + Au→ RS-Au + H+. Il CoA-CME può essere preparato per semplice immersione dell’elettrodo d’oro in una soluzione etanolica di coenzima. La modificazione è stata valutata tramite caratterizzazione della superficie valutando copertura superficiale, capacitanza del doppio strato e angolo di contatto. Si è verificato come Cu(II), Pb(II) e Cd(II) possano essere accumulati all’elettrodo modificato tramite preconcentrazione a circuito aperto (t=10 min) direttamente nel campione da analizzare. La concentrazione di metallo in soluzione è determinata successivamente in stripping a un potenziale al quale avviene la riduzione del metallo senza compromettere la stabilità del CoA-CME (Edep

=−400 mV, tdep = 60 s, Ei =−500 mV, Ef = +600 mV, v = 100 mV/s) operando in tampone citrato a pH 9 per evitare accumulo all’elettrodo. Le coppie Cu/Pb e Cu/Cd possono essere determinate simultaneamente mentre Cd/Pb, nelle condizioni sperimentali indicate, interferiscono avendo simile potenziale di picco. In condizioni tipiche si ottengono LOD di 0.3 per Cu, 0.8 ppb per Pb e 1.2 ppb per Cd. CME preparati con batch di CoA differenti hanno dato buone ripetibilità e riproducibilità. L’elettrodo modificato ha dimostrato avere maggiore affinità nei confronti del rame: a parità di concentrazione nel campione si ottengono correnti ca. 10 volte superiori rispetto a piombo e cadmio. Questi ultimi hanno sensibilità tra loro paragonabili. In campioni contenenti complessanti forti, quali ad es. EDTA, si ha depressione del segnale. Solo dopo titolazione completa del legante si registra il picco del metallo di intensità paragonabile a quella ottenuta in assenza di complessanti in soluzione: con questo CME solo una frazione di metallo labile può essere determinata nelle condizioni sperimentali indicate. La presenza di sostanza organica (acidi umici) riduce la sensibilità del metodo ma permette la determinazione del metallo totale: la presenza di Co-A attenua fenomeni di fouling dell’elettrodo. L’elettrodo modificato è stato applicato all’analisi di campioni di acque naturali a diversa salinità. Si possono ottenere anche informazioni sulla complessazione dei metalli nel campione considerato. I metalli sono stati determinati con le aggiunte standard ottenendo recuperi soddisfacenti (90%-103%). Riferimenti 1. W. Yang, J. J. Gooding, D. B. Hibbert, Journal of Electroanalytical Chemistry 516 (2001) 10. 2. R. K. Shervedani, S. A. Mozaffari, Surface & Coatings Technology 198 (2005) 123. 3. L. M.Niu, H. Q. Luo, N. B. Li, Arch. Pharm. Chem. Life Sci. 339 (2006) 356.

A BIOSENSOR FOR DETECTION OF ALL D-AMINO ACIDS

Elena ROSINI, Gianluca MOLLA, Silvia SACCHI, Stefano SERVI, Loredano POLLEGIONI

Centro Interuniversitario di Ricerca in Biotecnologie Proteiche “The Protein Factory”, Università degli studi dell’Insubria (DBSM, Varese) and Politecnico di Milano (DCMIC G. Natta, Milan), Italy

[email protected]

In food, D-amino acids (D-aa) are formed during processing and also originate from microbial sources, and may become part of the human diet. D-Ala, D-Asp and D-Glu are important constituents of the bacterial cell wall and peptide antibiotics and other metabolites produced by different micro-organisms also contain D-aa. Significant amounts of various D-aa have been found in milk and fermented dairy products. Both the total D-aa content and the D/(D+L) ratio have been proposed as reliable molecular markers of ripening and as indexes for quality assessment in these food products and thus considered indicators of product shelf-life. On the other hand, the presence of D-aa in non-fermented foods is considered an indicator of contamination of raw materials. A simple and accurate determination and quantification of D-aa in foods cannot be achieved using traditional methods (such as GC, CE and liquid HPLC), since these methods, which often are time-consuming and expensive, are usually not suitable for on-line application. For this reason, a biosensor as an analytical tool to measure the total content of D-aa appears as the best answer for setting up a fast, simple detection system with on-line capabilities. In past years, we proposed the use of the flavoenzyme D-amino acid oxidase (DAAO, EC 1.4.3.3, that catalyzes the oxidative deamination of D-aa into α-ketoacids, ammonia and hydrogen peroxide) to assay the D-aa concentration. Unlikely, the wild-type DAAO is inactive on acidic D-aa. Most recently we developed an amperometric biosensor that responds to all (neutral, acidic and basic) D-aa based on DAAO mutants (obtained by a directed evolution approach) displaying an evolved substrate specificity [1]. Due to the additive nature of the individual amino acid responses, this device can be used to sample solutions containing both single amino acid species and complex D-aa mixtures (Fig. 1). Figure 1. Average of the amperometric response of the enzymatic biosensors produced using immobilized wild-type and mutants of DAAO (values obtained on eight different 0.5 mM mixtures of D-aa) [1]. This procedure for determining the D-aa content is simple, rapid (10 minutes), and reliable. This assay requires a single electrode for each amperometric analysis (“one-shot” disposable device), with no need to regenerate the electrode surface. The device was successfully used to monitor the total D-aa content in a cheese sample: the amount of D-aa determined in Grana Padano cheese is in fairly good agreement with the value reported in the literature (∼6 mM). In conclusion, this electrochemical method compares favourably with standard methods for overall D-aa determination. References 1. E. Rosini, G. Molla, C. Rossetti, M.S. Pilone, L. Pollegioni, S. Sacchi, J Biotechnol. 135 (2008) 377.

DEPOSIZIONE DI FILM NANOSTRUTTURATI DI ZrO 2 PER APPLICAZIONI NEL CAMPO DELLA SENSORISTICA

Naida El Habra1,2, Marco Bolzan1, Chiara De Zorzi1, Andrea Sartori1, Monica Favaro1, Maurizio

Casarin2, Lioudmila Doubova3, Stefano Boldrini4, Gilberto Rossetto1

1 ICIS-CNR, Corsi Stati Uniti 4 - 35127 Padova, Italia, [email protected] 2Dipartimento di Scienze Chimiche, Università di Padova, Via Marzolo - 35131 Padova, Italia

3IENI-CNR, Corso Stati Uniti 4 – 35127 Padova, Italia 4ISIB-CNR, Corso Stati Uniti 4 – 35127 Padova, Italia

La zirconia (ZrO2) stabilizzata nella struttura cubica o tetragonale, mediante l’addizione di opportuni droganti di tipo MO e M2O3 (M = metallo aliovalente), è un materiale di considerevole interesse scientifico e tecnologico. Si tratta di un materiale refrattario resistente alle alte T, agli shock termici e manifesta, inoltre, un’elevata conduttività ionica. Queste caratteristiche sono tali da consentire un suo ampio impiego, non solo come materiale ceramico, ma anche come importante componente nelle fuel cells, in convertitori catalitici o nei sensori di gas. È noto che la zirconia stabilizzata con ossidi di metalli aliovalenti è essenzialmente un conduttore ionico la cui elevata conduttività è dovuta all’elevata concentrazione di vacanze di ossigeno create nel sottoreticolo anionico (1) in seguito alla sostituzione, nel network cationico, dello ione Zr4+ con cationi a valenza inferiore (M2+ e/o M3+). Dalla letteratura risulta che un’elevata concentrazione di vacanze di ossigeno costituisce un parametro importante ai fini applicativi della ZrO2 nel campo della sensoristica (2). La nanostrutturazione è un altro aspetto di considerevole importanza per incrementare le prestazioni del materiale, in quanto la presenza di grani su scala nanometrica ad elevata area superficiale specifica è tale da conferire al materiale un maggiore numero di siti attivi di superficie e perciò maggiori capacità di assorbimento rispetto ad un materiale ceramico, con conseguente incremento delle proprietà di ‘gas sensing’. Attualmente, la maggior parte dei sensori di gas commercialmente impiegati sono sensori elettrochimici a base di ZrO2 operanti ad alte T (700-900 °C). Numerosi tentativ i sono stati proposti per abbassare la loro T di operatività (2) sia mediante l’impiego di specifici trattamenti di superficie, sia preparando materiali alternativi in modo tale da avere elevata conduttività ionica a basse T, oppure impiegando film di elettrolita solido per ridurre l’impedenza totale della cella. Il processo CVD (Chemical Vapor Deposition) è una tecnica molto versatile che consente la deposizione di film solidi nanostrutturati, grazie alla possibilità di variare in modo mirato ed indipendente un elevato numero di parametri di processo sia chimici sia fisici. Nel presente lavoro si riportano i risultati ottenuti dalla deposizione di film densi e porosi nanostrutturati di ZrO2 stabilizzata nella fase cubica della fluorite (c-ZrO2) impiegando diversi ossidi di metalli aliovalenti (MgO, CaO, Sc2O3, Y2O3) e depositati mediante le tecniche MOCVD e Spray-Pyrolysis, rispettivamente. L’impiego dei due metodi di crescita ha consentito l’ottenimento di materiali con peculiari caratteristiche composizionali, morfologiche e microstrutturali. Le analisi composizionali (SIMS, EDS), morfologiche (FEG-SEM) e strutturali (XRD) dei materiali ottenuti, infatti, hanno confermato la deposizione di film solidi sia densi sia porosi di c-ZrO2 a seconda del metodo di deposizione impiegato. Al fine di verificare le loro proprietà di conducibilità elettrica, sono stati effettuati, inoltre, studi preliminari mediante misure di spettroscopia d’impedenza che hanno evidenziato valori di conducibilità dei materiali esaminati confrontabili con quelli riportati in letteratura (3) e tali da potersi ritenere dei buoni candidati per applicazioni nel campo della sensoristica. Ringraziamenti Tale lavoro di ricerca è supportato dai fondi del ‘Progetto FISR’ del Ministero dell’Istruzione e dell’Università della Ricerca Scientifica. Si ringrazia la FILA INDUSTRIA CHIMICA SPA per la messa a disposizione dello strumento FEG-ESEM FEI Quanta 200 e la Dr. C. Sada del Dipartimento di Fisica dell’Università di Padova per le analisi SIMS. Riferimenti 1. M.O. Zacate, L. Minervini, D.J. Bradfield, R.W. Grimes, K.E. Sickafus, Solid State Ion. 128 (2000) 243. 2. W. Cao, O.K. Tan, W. Zhu, B. Jiang, C.V. Gopal Reddy, Sens. Actuators B 77 (2001) 421, and

reference therein. 3. G. Meng, H. Song, Q. Dong, D. Peng, Solid State Ion. 175 (2004) 29.

COMPARISON OF DIFFERENT ANTIBODIES USED TO FABRICAT E NEW TRIAZINIC IMMUNOSENSORS AND AFFINITY CONSTANT V ALUES

Luigi Campanella1, Sergei Eremin2, Elisabetta Martini1, Mauro Tomassetti1

1 Department Chemistry of University “La Sapienza”, P.le a. Moro 5, 00185, Rome.

[email protected] 2Department of Chemical Enzymology, M. V. Lomonosov Moscow State University, Russia

Recently we developed a new immunosensor for the analysis of triazinic pesticides [1]. For this study we utilized an antibody produced by S. Eremin (Department of Chemical Enzymology of Moscow State University) named rabbit anti-atrazine (Lot. 7442 - Arina). In the present communication a comparison was made of analytical results recently obtained using as alternative another antibody named poly-IgG anti-atrazine (Lot. A9 - Arina), also produced by S. Eremin. The study was also extended to the ability of the antibody used and the analyte under test to give the immunocomplex. However the quality and characteristics of immunoreagent are of the great importance for all immunological methods. Theoretically a sensitive competitive immunoassay can be developed when the antibody affinity constant (Kaff) for the tracer (i.e. the marked antigen) is of the same order of magnitude as for the analyte. However, for a small antigen the affinity for the tracer is normally higher than for the analyte, as the antibody can recognise parts of the anchoring chain used for the hapten-carrier protein binding. Nevertheless, a rough estimation of the Kaff of the tracer antigen made by S. Eremin and al. [2] was found to be of the order of 2.4 x 108 M-1, while an estimation of Kaff of the different tracer used by us (where Kaff = 1/IC50) was found to be of the order of 0.95x108 M-1. In the first case the labeled immunogen used consisted of 1,3,5-triazine, substituted at position 2,4,6 (denoted as R1/R2/R3 groups) [2], that is NHiPr/S(CH2)2CONH-biotin-ALP/NHEt, while in the second case the immunogen was obtained by HRP labeling the triazine-BSA conjugated using the biotin – avidin method. In addition the affinity constant value of the antigen under test (i.e. atrazine) was also calculated at the midpoint of the calibration curves, obtained using different competition procedures. IC50 is the current at which 50% of the antibody complex was reached. The Kaff values were found to be of the order of 2.8x107 - 9.2x107 M-1. These values were compared with some Kaff values reported in literature by Eremin and several other authors using different immunoassay methods and was found to be in good agreement with the values found by us. Lastly a thorough investigation of selectivity values for the immunosensor developed was also carried out, both by studying several triazinic pesticides and considering pesticides of different classes (organophosphates, carbamates and organochlorides). References 1. L. Campanella, D. Lelo, E. Martini, M. Tomassetti. 1. Proceedings of the Sensors and Microsystems, XIII

Conferenza Annuale, Associazione Italiana Sensori e Microsistemi. 2. M. Franek, V. Kolar, S.A. Eremin, Analytica Chimica Acta 311 (1995) 349.

UN RIVELATORE AMPEROMETRICO BASATO SULL’USO DI LIQU IDI IONICI PER IL MONITORAGGIO IN FLUSSO DI NOX

Rosanna Toniolo, Andrea Pizzariello, Sabina Susmel, Nicolò Dossi, Gino Bontempelli

Dipartimento di Scienze e Tecnologie Chimiche, Università di Udine, via Cotonificio 108,

33100 Udine, [email protected]

Gli ossidi di azoto (NOx) sono specie gassose generate come sottoprodotti dei processi di combustione che come tali non sono in realtà dei veri e propri contaminanti atmosferici, in quanto solo raramente sono in grado di produrre direttamente effetti nocivi sulla salute. La loro pericolosità deriva invece dal fatto che essi, assieme agli idrocarburi volatili, sono gli ingredienti chiave nella formazione dello smog fotochimico che rappresenta una delle fonti più importanti di inquinamento ambientale [1].

L’esigenza di garantire un adeguata sicurezza a livello sia industriale che ambientale rende pertanto necessaria la disponibilità di semplici dispositivi di analisi, adatti al loro monitoraggio in situ ed in linea, in grado di proporsi quali valide alternative ai convenzionali approcci strumentali, come ad esempio le metodiche basate sulla chemiluminescenza, la gascromatografia, la cromatografia ionica e la spettrofotometria [2].

Questo obiettivo potrebbe essere facilmente raggiunto facendo ricorso alle tecniche elettroanalitiche, in vista dei vantaggi da esse offerte in termini sia di economicità della strumentazione necessaria che di facile automatizzazione e trasduzione dei segnali con esse acquisiti. Tuttavia, la loro utilizzazione per il monitoraggio di NOx è ostacolata dalla concomitante presenza nelle atmosfere da sottoporre a monitoraggio di biossido di zolfo, il quale è anch’esso un contaminante suscettibile di ossidazione elettronica, portando quindi a determinazioni scarsamente selettive [3]. Nel corso di indagini sistematiche attualmente svolte allo scopo di caratterizzare le proprietà elettrochimiche dei liquidi ionici (ILs, sali a punto di fusione molto prossimi alla temperatura ambiente, costituiti da cationi e anioni di natura organica, i quali agiscono da solventi ionici caratterizzati da bassa tensione di vapore, alta viscosità, buona stabilità termica, elevata conducibilità elettrica e capacità solvente ampiamente modulabile) è stato possibile osservare che in alcuni di tali mezzi il processo di ossidazione di SO2 ad elettrodi di Pt risulta essere affetto da notevole sovratensione, così da decorrere a potenziali molto più elevati di quelli necessari per l’ossidazione di NOx. Questo ha suggerito la possibilità di adottare una sonda elettroanalitica per gas basata sull’impiego di ILs, precedentemente da noi sviluppata [4], modificandola opportunamente per renderla adatta al monitoraggio di NOx.

Tale sonda è costituita da tre elettrodi (due di Pt ed uno di Ag) che fuoriescono per qualche frazione di mm da una superficie in Teflon e che vengono messi in contatto elettrolitico tra loro ricoprendo la superficie di Teflon con uno strato sottile di IL. Il liquido ionico utilizzato era l’ 1-butil-3-metilimidazoliobis(trifluorometansulfonil) imide (BMIM-NTF2), scelto come più adatto sulla base dei risultati ottenuti valutando il comportamento voltammetrico di NOx e SO2 in una serie di ILs diversi. E’ stato così possibile sviluppare un sensore amperometrico per la determinazione selettiva in flusso per gli NOx a livello di ppb, anche in presenza di SO2 senza che sia necessario ricorrere ad alcun stadio di preconcentrazione o all’uso di filtri-trappola per la rimozione dell’interferente e capace di operare in un intervallo di temperatura compreso tra ca. 20 e 100°C. Riferimenti 1. Health effects of transport-related air pollution Eds. M. Krzyzanowsky, B. Kuna-Dibbert, J. Schneither, 1st

Edition WHO 2005. 2. P. Jacquinot, A.W.E. Hodgson, P.C. Hauser, Anal. Chim. Acta 443 (2001) 53. 3. I. Bergman, J. Electroanal. Chem. 157 (1983) 59. 4. R. Toniolo, A. Pizzariello, S. Susmel, N. Dossi, A.P. Doherty, G. Bontempelli, Electroanal. 19 (2007)

2141.

DETECTION OF TYPE A-TRICHOTHECENES IN BREAKFAST CER EALS AND CEREAL-BASED BABY FOODS USING A NOVEL ELECTROCHEMIC AL

IMMUNOSENSOR METHOD

Silvia Vesco, Daniela Romanazzo, Francesco Ricci, Giulia Volpe, Danila Moscone, Giuseppe Palleschi

Dipartimento di Scienze e Tecnologie Chimiche,

Università di Roma Tor Vergata, Via della Ricerca Scientifica, 00133, Roma [email protected]

Within the EC-funded integrated project BioCop , an electrochemical immunosensor method was developed for the determination of T-2 and HT-2 toxins in cereals and cereal-based foods. A test portion is extracted with acetonitrile/water (86/14). The sample extract is centrifuged and purified, using a Mycosep column, and dried under nitrogen flow. The residue is resuspended and diluted in PBS buffer at the moment of the analysis. The total amount of HT-2 and T-2 toxins is quantified by a competitive ELIME-array (Enzyme-Linked-Immuno-Magnetic-Electrochemical-array) method. The method was in-house validated and yielded the following recovery characteristics. For breakfast cereals: Recovery for sample spiked with T-2/HT-2 toxins at 200 ng/g: 104 %; Recovery for test portion naturally contaminated with T-2/HT-2 toxins at 80 ng/g sample: 125 %. For maize-based baby foods: Recovery for sample spiked with T-2/HT-2 toxins at 20 ng/g: 106 %; Recovery for test portion naturally contaminated with T-2/HT-2 toxins at 21 ng/g sample: 123 %. Matrix effects hardly occurred. Currently the method is undergoing interlaboratory pre-validation to establish preliminary within- and between-laboratory precision data in several BioCop-partner laboratories. A full collaborative study is planned to be undertaken in 2010.

NANOSTRUCTURED SURFACES FOR AMPEROMETRIC BIOSENSORS

Chiara Zanardi1, Luisella Brunzu1, Clara Bandoli2, Emanuela Licandro2, Stefano Maiorana2, Patrizia R. Mussini3, Laura Pigani1, Fabio Terzi1, Barbara Zanfrognini1, Renato Seeber1

1 Dipartimento di Chimica, Università di Modena e Reggio Emilia, via G.Campi 183,

[email protected] 2Dipartimento di Chimica Organica ed Industriale, Università degli Studi di Milano, via Venezian 21

3Dipartimento di Chimica Fisica ed Elettrochimica, Università degli Studi di Milano, via Golgi 19

Electrochemical biosensing is significantly improving in the last years, thanks to the development of different enzyme biosensors, immunosensors, and DNA sensors. Many novel devices have been successfully proposed for clinical, food, and environmental analyses. One of the most critical steps toward the construction of a biosensor lies in anchoring the recognition element on the transducer, which has to be suitably chosen in order to stably fix biological elements on a metal surface without detrimental effect on the biological activity. The possibility to use quick deposition methods is often considered an added value. In this communication we discuss the effectiveness of Au nanostructured surfaces in amperometric sensors, in order to achieve stable anchoring of a number of individual biological recognition elements larger than in the case of conventional planar Au electrodes. The increase of the number of adsorbed molecules, in turn, leads to an increase the electrochemical signal. Au nanoparticles differently anchored on Au substrates constitute the surface nanostructures developed; in addition, Au nanostructures are generated directly on planar Au electrodes, taking advantage of suitable roughening electrochemical procedures. Literature reports different strategies for the anchoring of the biological recognition elements on Au surfaces. In many cases, the biological molecules are suitably functionalized, e.g. with a thiol terminal group, in order to be stably adsorbed on Au. In the present communication, we check the capability of amino terminal groups in achieving stable interaction with our nanostructured Au surfaces. To such an extent, we used amino derivatives bearing a ferrocene electroactive moiety as a probe. The stability of the adsorbed molecules on the surfaces and the reproducibility of the deposition procedures have been checked using electrochemical techniques, by exploiting the signal relative to ferrocene reversible oxidation. The advantages of the use of different nanostructured surfaces with respect to bare Au, in terms of sensitivity of the amperometric signal, have been ascertained. Furthermore, the above described nanostructured surfaces were tested for developing DNA sensors based on peptide nucleic acids (PNA) as recognition elements. In this case, the possibility to use a simple amino group as anchoring moiety, with respect to the commonly used thiol group, should be preferred, since the synthesis of the relevant PNA derivatives results easier.

SYNTHESIS AND CHARACTERISATION OF [Co(2,2’:6’,2’’-t erpyridine) 2]2+-BASED METALLOPOLYMERS

Chiara Zanardia, Barbara Zanfrogninia, Fabio Terzia, Laura Pigania, Renato Seebera, Stefania

Morandia, Chiara Danielia, Luca Pasqualib, Monica Montecchib, Stefano Nannaroneb

aDipartimento di Chimica, Università di Modena e Reggio Emilia [email protected]

bDipartimento di Ingegneria dei Materiali e dell’Ambiente, Università di Modena e Reggio Emilia Conducting polymers constitute a class of materials well recognised as interesting electrode coatings for the development of amperometric sensors. The insertion into the polymeric matrix of metal ions suitable to be reversibly oxidised and/or reduced, constitutes a potentially powerful way to further increase the electrocatalytic properties of the whole material. The resulting hybrid materials are generally known as ‘conducting metallopolymers’, which combine the conductivity of conducting polymers with the redox and optical properties of coordination complexes. Our attention is particularly devoted to polythiophene derivatives bearing coordinating side ligands for metal ions such as those of VIII group. In this context, we have recently synthesised two derivatives of a terthienyl-substituted 2,2’:6’,2’’-terpyridine. On the one hand, the oligothiophene chain allows the formation of a polymeric coating under very mild oxidative conditions; on the other hand, the terpyridine moiety is suitable to bond different metal ions, such as Co(II), but also Ru(II), Ir(II) and Ni(II).

Co2+

S S S

N

N

N

O O

SSS

N

N

N

OO

1

Co2+

S S S

N

N

N

S

S

SSS

N

N

N

S

S

2

In this work we report the first investigations on complexes 1 and 2, containing Co(II) centres. The two complexes have been characterised through different techniques, i.e. NMR and UV-Vis spectroscopies, and different electrochemical methods. They can be very easily electropolymerised on Pt surfaces, resulting in well stable electrode coatings. The voltammograms of the two hybrid materials are characterised by reversible peak systems associated to Co(II) and to the oligothienyl moiety. Spectroelectrochemical studies on ITO glass have been performed in order to more deeply investigate the charge and discharge processes of the film. The oxidation state of the metal centres and the electronic properties of the organic moiety have been defined through photoemission spectroscopies employing X-Ray and UV radiations. These measurements have been complemented by Near Edge X-Ray Absorption Fine Structure (NEXAFS) investigations.

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