Bioanalitica 2019
“From molecules to devices”
6 Dicembre 2019
Centro Congressi S. Elisabetta
Parco Area delle Scienze 95, Campus Universitario, Parma
ISBN 978-88-94952-14-8 © Società Chimica Italiana 2019
Edited by Mara Mirasoli and Massimo Guardigli Published online on 6 December 2019 at Parma (Italy)
ORGANIZZAZIONE
COMITATO SCIENTIFICO
Mara Mirasoli (Alma Mater Studiorum - Università di Bologna) Chiara Cavaliere (Sapienza - Università di Roma)
Claudio Baggiani (Università di Torino) Ilaria Palchetti (Università di Firenze)
Maria Careri (Università di Parma) Scriba Nanotecnologie S.r.l.
Gruppo Bioanalitica
Società Chimica Italiana
Divisione Chimica Analitica
Il Gruppo di Bioanalitica della Divisione di Chimica Analitica della Società Chimica Italiana riunisce ricercatori che, nei settori accademico, istituzionale e industriale, si occupano dello sviluppo di metodi bioanalitici o utilizzano tali metodi per scopi di ricerca o applicativi.
Il Gruppo di Bioanalitica organizza, a cadenza annuale, giornate scientifiche che hanno lo scopo di riunire gli aderenti per discutere tematiche di interesse, attuali o emergenti, promuovendo in tal modo il confronto e la collaborazione scientifica.
La giornata Bioanalitica 2019 è un evento congiunto con il Workshop "From molecules to devices" organizzato dalla Prof.ssa Mariella Careri (Dipartimento di Scienze Chimiche, della Vita e della Sostenibilità Ambientale dell'Università di Parma) nell'ambito del progetto del Dipartimento di eccellenza.
La giornata è dedicata alla collega e amica Cristina Giovannoli, Prof.ssa di Chimica Analitica all'Università degli Studi di Torino, prematuramente scomparsa pochi mesi fa.
Programma
9:00
Registrazione
Sessione comune con il Workshop “From molecules to devices”
Auditorium
9:15
Saluti e introduzione ai lavori
Nanotechnologies for sensing and devices (Chair: W. Knoll)
9:30 Plenary lecture
PL SILICA NANOPARTICLES AS LUMINESCENT PLATFORMS FOR IMAGING AND
SENSING APPLICATIONS L. Prodi
Department of Chemistry “G. Ciamician”, Alma Mater Studiorum - University of Bologna, Bologna (Italy)
10:10 Keynote Lecture
KN1 DNA-BASED NANODEVICES FOR DIAGNOSTIC APPLICATIONS
F. Ricci
Chemistry Department, University of Rome Tor Vergata, Rome (Italy)
10:40
Coffee break e sessione poster
Sessione Giornata Bioanalitica 2019
Aula Master
Introduction and Prizes (Chair: M. Mirasoli)
11:00 RICORDO DI CRISTINA GIOVANNOLI M. Vincenti
Department of Chemistry, University of Turin, Turin (Italy
CONSEGNA DEL PREMIO “GIOVANE RICERCATORE” DI BIOANALITICA AL DOTT. FABIO DI NARDO DELL’UNIVERSITÀ DI TORINO
11:25 Keynote Lecture del vincitore del premio “Giovane Ricercatore” di Bioanalitica KN2 RECENT ADVANCES AND FUTURE PERSPECTIVES IN LATERAL FLOW
ASSAY F. Di Nardo
Department of Chemistry, University of Turin, Turin (Italy)
Bioanalytical mass spectrometry and separation science; Drug development
(Chair: Marco Vincenti)
11:45 O1 DEVELOPMENT OF A NEW INTEGRATED ANALYTICAL PLATFORM FOR THE
IDENTIFICATION OF SHORT PEPTIDES IN COW MILK
C.M. Montonea, S.E. Aitaa, A.L. Capriottia, M. Antonellia, A. Cerratoa, S. Piovesanaa, A. Laganàa,b
a
Department of Chemistry, Università di Roma “La Sapienza”, Rome (Italy)
bCNR NANOTEC, Campus Ecotekne, University of Salento, Lecce (Italy)
12:00 O2 HELICAL MULTI-WALLED CARBON NANOTUBES COATED FIBERS FOR SPME
DETERMINATION OF POLYCYCLIC AROMATIC HYDROCARBONS AT ULTRA-TRACE LEVELS IN ICE CORES
A. Arcoleo, F. Bianchi, M. Careri
Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, Parma (Italy)
12:15 O3 EXPLORING THE UNDISCLOSED HEMP COMPOUNDS: THE COMPREHENSIVE POLAR LIPIDOME TOOL FOR THE ENRICHMENT, FRACTIONATION AND IDENTIFICATION
M. Antonellia, S.E. Aitaa, B. Benedettia, C. Cavalierea, A. Cerratoa, A. Laganàa,b
aDepartment of Chemistry, Università di Roma “La Sapienza”, Rome (Italy) b
CNR NANOTEC, Campus Ecotekne, University of Salento, Lecce (Italy)
12:30 O4 DRUG DEVELOPMENT AND LOADING STUDY OF POLYPEPTIDE-DRUG
CONJUGATES THROUGH ASYMMETRICAL FLOW FIELD-FLOW FRACTIONATION – MULTIDETECTION
V. Marassia,b, J.J. Arroyoc, M.J. Vicentc, B. Rodaa,b, P. Reschigliana,b, A. Zattonia,b
a
Department of Chemistry “G. Ciamician”, University of Bologna, Bologna, (Italy)
b
byFlow Srl, Bologna, Italy
cPolymer Therapeutics Lab. Príncipe Felipe Research Institute, Valencia (Spain)
12:45 O5 3D PRINTED ALGINATE/NANOCRYSTALLINE CELLULOSE-BASED SCAFFOLDS
LOADED WITH SILVER NANOPARTICLES COMBINING ANTIMICROBIAL AND CYTOTOXIC EFFECTS TOWARDS HepG2 CELL LINE
C. Bergonzia, S. Marandoa, L. Bergamontib, C. Graiffb, M.C. Ossiprandic, I. Zanottia, F. Berninia, R. Bettinia, L. Elviria
aFood and Drug Department, University of Parma, Parma (Italy) b
Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, Parma (Italy).
c
13:00
Lunch break e sessione poster
Paper-based assays and cell-based assays (Chair: Giuseppe Palleschi)
13:55 O6 A PAPER-BASED ELECTROCHEMICAL DEVICE FOR THE DETECTION OF
PESTICIDES INSPIRED BY NATURE: A FLOWER-LIKE ORIGAMI BIOSENSOR F. Arduini, V. Caratelli, G. Fegatelli, G. Ciampaglia, G. Palleschi, D. Moscone Department of Chemical Science and Technologies, University of Rome Tor Vergata, Rome (Italy)
14:10 O7 A RAPID AND SENSITIVE LATERAL FLOW IMMUNOASSAY FOR THE REAL
TIME MONITORING OF THE COMPLIANCE IN ANTIRETROVIRAL THERAPIES: FROM HAPTEN SYNTHESIS TO PROTOTYPE
S. Cavaleraa, F. Di Nardoa, L. Anfossia, C. Baggiania, M. Simieleb, A. D'Avoliob, J.V. Mercaderc, A. Abad-Fuentesc, C. Agullόd, A. Abad-Somovillad
a
University of Torino, Department of Chemistry, Torino (Italy)
bCoQuaLab, University of Torino, Department of Medical Sciences, Torino (Italy) c
CSIC, Instituto de Agroquímica y Tecnologia de Aliméntos, Paterna, Valencia (Spain)
d
Universitat de Valencia, Facultat de Química Organica, Paterna, Valencia (Spain)
14:25 O8 ORIGAMI PAPER-BASED SENSORS AND PRECISION MEDICINE: AN
APPLICATION IN ALZHEIMER DISEASE
V. Caratelli, A. Ciampaglia, J. Guiducci, D. Moscone, F. Arduini
Department of Chemical Science and Technologies, University of Rome Tor Vergata, Rome (Italy)
14:40 O9 A MULTIPLEX LFIA BIOSENSOR FOR THE ON-FIELD IDENTIFICATION AND
SEROTYPING OF CATTLE FOOT-AND-MOUSE DISEASE VIRUS
L. Anfossia, A. Russoa, S. Graziolib, G. Pezzonib, C. Nogarolc, F. Di Nardoa, S. Cavaleraa, C. Baggiania, S. Rosatid, E. Brocchib
a
Department of Chemistry, University of Turin, Torino (Italy)
b
Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna, Brescia (Italy)
cIn3diagnostic, s.r.l. Grugliasco (TO, Italy) d
Department of Veterinary Science, University of Turin, Grugliasco (TO, Italy)
14:55 O10 NANO-LANTERN ON PAPER FOR SMARTPHONE-BASED ATP DETECTION
M.M. Calabrettaa, E. Michelinia,b, R. Álvarez-Didukc, L. Montalia, A. Lopresidea, A. Merkoçic,d, A. Rodaa,b
a
Department of Chemistry “G. Ciamician”, University of Bologna, Bologna, (Italy)
b
INBB, Istituto Nazionale di Biostrutture e Biosistemi, Roma, (Italy)
c
ICN2 - Nanobioelectronics & Biosensors Group, Institut Catala de Nanociencia i Nanotecnologia, Campus UAB, Bellaterra, Barcelona (Spain)
d
ICREA - Institució Catalana de Recerca i Estudis Avançats, Barcelona (Spain)
15:10 O11 BIOACTIVE MOLECULES CHARACTERIZATION BY HPLC-MS/MS AND
BIOLOGICAL EVALUATION OF NADES EXTRACTS FROM WINEWASTE AND ITS POTENTIAL USE IN COSMETIC FIELD
A. Punzoa, E. Porrua, D. Calabriaa, M. Mirasolia, M. Guardiglia, C. Samorìa, P. Gallettia, C. Calicetib, E. Tagliavinia, A. Rodaa
aDepartment of Chemistry “G. Ciamician”, University of Bologna, Bologna (Italy) b
15:25
Coffee break e sessione poster
Sensors and biosensors (Chair: Marco Giannetto)
15:50 O12 AN ENZYME-LINKED DNA-BASED ARRAY FOR THE ELECTROCHEMICAL
DETECTION OF AFLATOXIN B1
G. Selvolinia, M. Lettieria, G. Marrazzaa,b
a
Department of Chemistry “Ugo Schiff”, University of Florence, Sesto Fiorentino (FI), Italy
bINBB – National Institute of Biostructures and Biosystems, Rome (Italy)
16:05 O13 A NANOHYBRID BIMETALLIC PLASMONIC ACTIVE PLATFORM FOR
HYDROGEN PEROXIDE SENSING AND GLUCOSE BIOSENSING
A. Scroccarelloa, F. Della Pellea, E. Fratinib.c, G. Ferrarob,c, S. Scaranob, D. Compagnonea
aFaculty of Bioscience and Technology for Food, Agriculture and Environment, University of
Teramo, Campus “Aurelio Saliceti”, Teramo (Italy)
b
Department of Chemistry “Ugo Schiff”, University of Florence, Sesto Fiorentino (Italy)
c
Center for Colloid and Surface Science (CSGI), University of Florence, Sesto Fiorentino (Italy) 16:20 O14 GRAPHENE OXIDE DERIVATIVES FOR WEARABLE SENSORS
F. Polettia, L. Favarettob, A. Kovtunb, E. Treossib, V. Palermob,c, M. Meluccib, C. Zanardia,b
aDepartment of Chemical and Geological Sciences, University of Modena and Reggio Emilia,
Modena (Italy) b
Institute of Organic Synthesis and Photoreactivity, National Research Council of Italy (CNR), Bologna (Italy)
c
Industrial and Materials Science, Chalmers University of Technology, Göteborg (Sweden) 16:35 O15 MICROBUBBLE RESONATOR-BASED OPTICAL IMMUNOSENSOR FOR
MONITORING EMERGING CONTAMINANTS IN WATERS F. Bettazzia, A. Giannettib, S. Berneschib, I. Palchettia
a
Chemistry Department, University of Florence, Sesto Fiorentino (FI), Italy
b
CNR-IFAC “Nello Carrara” Institute of Applied Physics, Sesto Fiorentino (FI), Italy
Poster
P1 CORTISOL, TESTOSTERONE AND TRUST: THE CONTRIBUTION OF PHYSIOLOGICAL
MEASUREMENTS TO A BEHAVIORAL STUDY AIMED AT UNDERSTANDING HOW THE REPRESENTATIONS OF THE OTHERS AFFECT TRUST
L. Anfossia, P. Conzob, C.O. Mossoc, F. Spaccatinid, F. Di Nardoa, S. Cavaleraa, C. Baggiania
a
Department of Chemistry, University of Turin, Torino
b
Department of Economics & Statistics "Cognetti de Martiis”, University of Turin, Torino
cDepartment of Psychology, University of Turin, Torino d
Department of Institutions and Society, University of Perugia, Perugia
P2 PREPARATION OF DECELLULARIZED RAT THYROIDS FOR TISSUE ENGINEERING
APPLICATIONS: A MASS SPECTROMETRY CHARACTERIZATION
F. Barbaroa, G. Di Conzaa, E. Consolinia, M. Alfieria, D. Dallatanaa, C. Bergonzib, G. Remaggib, R. Bettinib, R. Tonia, L. Elvirib
a
Department of Medicine and Surgery, University of Parma, Parma, Italy
bFood and Drug Department, University of Parma, Parma, Italy
P3 METAPROTEOMIC INVESTIGATION OF BIOAEROSOL FROM WORK ENVIRONMENTS
A.L. Capriotti, C.M. Montone
Department of Chemistry, University of Rome “La Sapienza”, Rome (Italy)
P4 A SMART AND PREDICTIVE WHOLE-CELL BIOANALYTICAL METHOD BASED ON TURN-ON
CHEMILUMINESCENCE DIOXETANE PROBE SENSING TO QUANTIFY INTRACELLULAR H2O2 AND FOR ANTIOXIDANT SCREENING IN HUMAN LIVING CELL
D. Calabriaa, M. Guardiglia, M. Mirasolia, P. Simonib, M. Zangheria, E. Pagnottac, A. Punzoa, C. Calicetid,e, A. Rodaa,e
a
Department of Chemistry “Giacomo Ciamician”, University of Bologna, Bologna (Italy)
b
Department of Medical and Surgical Sciences, University of Bologna, Bologna (Italy)
cCouncil for Agricultural Research and Economics, Research Centre for Cereal and Industrial Crops, Bologna
(Italy)
d
Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna (Italy)
eInterdepartmental Centre of Industrial Agrifood Research (CIRI Agrifood), University of Bologna, Cesena, (Italy)
P5 CHARACTERIZATION OF POLAR LIPIDOME OF SPIRULINA MICROALGAE BY AN OPTIMIZED
LIQUID CHROMATOGRAPHY SEPARATION AND HIGH-RESOLUTION MASS SPECTROMETRY C. Cavalierea, C.M. Montonea, M. Antonellia, A. Cerratoa, S.E. Aitaa, B. Benedettia, A. Laganàa,b
aDepartment of Chemistry, Università di Roma “La Sapienza”, Rome, Italy b
CNR NANOTEC, Campus Ecotekne, University of Salento, Lecce, Italy
P6 GRAPHITIZED CARBON BLACK ENRICHMENT PROCEDURE FOR THE DETECTION OF
PHOSPHOLIPIDS IN OLIVE OIL BY LIQUID CHROMATOGRAPHY-HIGH RESOLUTION MASS SPECTROMETRY
aDepartment of Chemistry, Università di Roma “La Sapienza”, Rome, Italy b
CNR NANOTEC, Campus Ecotekne, University of Salento, Lecce, Italy
P7 PAPER BLUE: AN EFFECTIVE TOOL FOR ELECTROCHEMICAL AND COLORIMETRIC POC
TESTING
S. Cintia, N. Bagherib,c, V. Caratellib, R. Massoudd, D. Mosconeb, G. Palleschib, F. Arduinib
a
Department of Pharmacy, University of Naples “Federico II”, Naples (Italy)
bDepartment of Chemical Science and Technologies, University of Rome “Tor Vergata”, Rome (Italy) c
Department of Chemistry, Isfahan University of Technology, Isfahan (Iran)
d
Department of Experimental Medicine and Surgery, University of Rome “Tor Vergata”, Rome (Italy)
P8 COMPUTATIONAL APPROACH FOR THE DETERMINATION OF NPS METABOLISM BY
MEANS UHPLC-HRMS
F. Fantia, F. Vincentib,c, C. Montesanob, E. Olivaa, A. Gregorid, A.R. Tognae, M. Martif, D. Compagnonea, M. Sergia
a
University of Teramo, Faculty of Bioscience and Technology for Food, Agriculture and Environment, Teramo, Italy
b
Sapienza University of Rome, Department of Chemistry, Rome, Italy
c
Sapienza University of Rome, Department of Public Health and Infectious Disease, Rome, Italy
dCarabinieri, Department of Scientific Investigation (RIS), Rome, Italy e
Sapienza University of Rome, Department of Physiology and Pharmacology Vittorio Erspamer, Rome, Italy
f
University of Ferrara, Department of Life Sciences and Biotechnology (SVeB), Ferrara, Italy
P9 STAND-ALONE PLASTIC ELECTRODE BASED ON PHOSPHOMOLYBDATE ELECTROACTIVE
COMPLEX FOR PHOSPHATE DETECTION IN ENVIRONMENTAL SAMPLES
F. Figueredoa, M. Lekkab, R. Offoiachb, A. Annibaldic, F. Girolamettic, S. Susmela
a
Department of Agricultural, Food, Environmental and Animal Sciences-Di4A, University of Udine, Udine (Italy)
bPolytechnic Department of Engineering and Architecture-DPIA, University of Udine, Udine (Italy) c
Department of Life and Environmental Sciences, Università Politecnica delle Marche, Ancona, (Italy)
P10 CHITOSAN-BASED BIOMATERIALS FOR CHONDROGENESIS ENHANCEMENT AND
MS-PROTEOMICS INVESTIGATION ON THEIR DEDIFFERENTIATION PROCESS
S. Marandoa, C. Bergonzia, E. De Angelisb, A. Cattaneoc, P. Borghettib, R. Bettinia, A. Bachic, L. Elviria
a
Food and Drug Department, University of Parma, Parma, Italy
bDepartment of Veterinary Science, University of Parma, Parma, Italy c
IFOM-FIRC Institute of Molecular Oncology, Milan, Italy
P11 CHEMILUMINESCENT BASED STIMULUS-RESPONSIVE BREAKABLE ORGANOSILICA
NANOCAPSULES
E. Marchegiania, M.D.C. Ortega Liebanab, M. Mirasolia, A. Rodaa, L. De Colab
a
Dipartimento di Chimica “G. Ciamician”, University of Bologna, Bologna, Italy
b
Laboratoire de Chimie et des Biomatériaux Supramoléculaires Institut de Science et d'Ingénierie Supramoléculaires, Université de Strasbourg & CNRS, Strasbourg, France
P12 MINIATURIZED AND USER-FRIENDLY BIOSENSOR DEVELOPING: A LAB-ON-A-TIP
APPROACH TO PERFORM FAST AND POINT-OF-CARE ANALYSIS OF GLUCOSE IN DRINKS V. Mazzaracchioa, S. Cintib, R. Marronea, D. Mosconea, F. Arduinia
a
Department of Chemical Science and Technology, University of Rome “Tor Vergata”, Rome, Italy
b
P13 COMPARISON OF ELECTROCHEMICAL AND SPR TRANSDUCTION IN SENSORS WITH A
MOLECULARLY IMPRINTED POLYMER AS BIOMIMETIC RECEPTOR M. Pesaventoa, A. Profumoa, R. Biesuza, N. Cennamob, L. Zenib
aDepartment of Chemistry, University of Pavia (Italy) b
Department of Engineering, University of Campania “Luigi Vanvitelli“, Aversa (Italy)
P14 SYNTETHIC OPIOIDS: TARGET AND UNTARGET ANALYSIS OF FENTANYL DERIVATIVES IN
ORAL FLUID
F. Vincentia,b, C. Montesanoa, S. Piraua, A. Gregoric, M. Sergid, R. Curinia
a
Department of Chemistry, Sapienza University of Rome, Rome, Italy
bDepartment of Public Health and Infectious Disease, Sapienza University of Rome, Rome, Italy cCarabinieri, Department of Scientific Investigation (RIS), Rome, Italy
d
Faculty of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, Teramo, Italy
P15 AN IOT PORTABLE ELECTROCHEMICAL IMMUNOSENSOR FOR DETERMINATION OF
HUMAN EPIDIDYMIS PROTEIN 4 AS A BIOMARKER FOR OVARIAN CANCER M. Mattarozzia, V. Bianchib, M. Giannettoa, I. De Munarib, A. Bonib, M. Careria
aDepartment of Chemistry, Life Sciences and Environmental Sustainability, Parma (Italy) b
Department of Engineering and Architecture, University of Parma, Parma (Italy)
P16 ELECTROCHEMILUMINESCENCE MEETS NANOTECHNOLOGY: NANOMATERIALS FOR
ENHANCE THE SIGNALS
S. Rebeccania, A. Zanuta, F. Arcudib, E. Rampazzoa, G. Valentia, M. Pratob, L. Prodia, F. Paoluccia
a
Department of Chemistry “Giacomo Ciamician”, University of Bologna, Bologna (Italy)
b
SESSIONE COMUNE
CON IL WORKSHOP
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Woorrkksshhoopp““FFrroommmmoolleeccuulleessttooddeevviicceess””::
Dipartimento di Scienze Chimiche, della Vita e della Sostenibilità Ambientale, Università di Parma
B
Biiooaannaalliittiiccaa2200119 9
Gruppo Divisionale Bioanalitca, Divisione di Chimica Analitica, Società Chimica Italiana 6 Dicembre 2019, Centro Congressi S. Elisabetta (Parma)
Silica Nanoparticles as luminescent platforms for imaging and sensing
applications
Luca Prodi
Department of Chemistry “Giacomo Ciamician”, University of Bologna, Bologna (Italy) e-mail: luca.prodi@unibo.it
Silica nanoparticles are versatile platforms with many intrinsic features, including a low toxicity. Proper design and derivatization yield particularly stable, very bright nanosystems displaying multiple functions [1], which can be used for either otpical and photoacoustic imaging [2] and for photoluminescence (PL)[3] and electrochemi-luminescence (ECL) sensing [4]. In addition, silica nanoparticles can also be used for as platforms for photo-dynamic and photo-thermal therapies.2 For these reasons, silica nanoparticles already offer unique opportunities, and further improvement and optimization can substantially expand their possible applications in fields of high impact, such as medical diagnostics and therapy, environmental and food analysis, and security.
In this context, we have developed a direct micelle assisted strategy based on the use of Pluronic F127 as high molecular weight surfactants. The one-pot synthesis yields PEGylated silica nanoparticles endowed with very high monodispersity, colloidal stability and core-shell structure. These nanoparticles were recently reported with the acronym PluS NPs (Pluronic Silica NanoParticles). These NPs had a silica core of about 10 nm and an overall hydrodynamic diameter of about 25 nm. Interestingly, PluS NPs can be tailored for optimization of processes such as directional energy transfer, which provide those systems with extremely valuable functions: high light-harvesting capability, signal-to-noise maximization, multiplex output, and signal amplification. In-vivo experiment proved the absence of toxic effects.
We will focus our presentation to the use of these systems for imaging and sensing applications, such as the possibility to monitor the presence of illicit drugs.4
Figure 1: Sensing mechanism for the detection of ecstasy [4].
References
[1] M. Montalti, L. Prodi, E. Rampazzo, N. Zaccheroni, “Dye Doped Silica Nanoparticles as Luminescent Organized Systems for Nanomedicine”, Chem. Soc. Rev., 2014, 43, 4243.
[2] S. Biffi, L. Petrizza, C. Garrovo, E. Rampazzo, L. Andolfi, P. Giustetto, I. Nicolov, G. Kurdi, M. Danailov, G. Zauli, P. Secchiero, L. Prodi, “Multimodal NIR-Emitting PluS Silica Nanoparticles with Fluorescent, Photoacoustic and Photothermal Capabilities”, Int. J. Nanomedicine, 2016, 11, 4865.
[3] D. Masseroni, E. Biavardi, D. Genovese, E. Rampazzo, L. Prodi, E. Dalcanale, “A fluorescent probe for ecstasy”, Chem. Commun., 12799-12802 (2015).
[4] G. Valenti, E. Rampazzo, E. Biavardi, E. Villani, G. Fracasso, M. Marcaccio, F. Bertani, D. Ramaldi, E. Dalcanale, F. Paolucci, L. Prodi, “An electrochemiluminescent-supramolecular approach to sarcosine detection for early diagnosis of prostate cancer”, Faraday Discuss., 2015, 185, 299-309.
Workshop “From molecules to devices” Workshop “From molecules to devices” : :
Dipartimento di Scienze Chimiche, della Vita e della Sostenibilità Ambientale, Università di Parma B
B ioanalitica ioanalitica 20192019
Gruppo Divisionale Bioanalitca, Divisione di Chimica Analitica, Società Chimica Italiana 6 Dicembre 2019, Centro Congressi S. Elisabetta (Parma)
DNA-based nanodevices for diagnostic applications
Francesco RicciChemistry Department, University of Rome, Tor Vergata, Rome, Italy email: francesco.ricci@uniroma2.it
Nature has invented a number of tricks and strategies by which the behaviour of proteins and other biomolecular machines can be finely controlled. These highly optimized and evolved mechanisms allow to control biological pathways with different chemical and environmental stimuli and are at the basis of the high specificity and selectivity of biomolecular machines. Motivated by the above arguments we have characterized and recreated in-vitro several mechanisms to control the response of DNA-based nanodevices for diagnostic and drug-delivery applications. Using these mechanisms we can finely control the activity of DNA-based nanodevices with different chemical and environmental stimuli including pH, antibodies, enzymes, small molecules and electronic inputs.
I will present an overview of the most representative and recent examples developed in our lab in the above research directions focusing on examples of DNA-based nanodevices for diagnostic applications.
LETTURA PREMIO
Recent advances and future perspectives in Lateral Flow Assay
Fabio Di Nardo
Department of Chemistry, University of Torino, Torino (Italy) e-mail: fabio.dinardo@unito.it
Standard analytical methods have high accuracy and precision, and are usually used for quantitative analysis. They often require expensive equipment and professional expertise, and may take hours or days to obtain results. However, in clinical, drug, food and environmental analysis it is often required to evaluate the content of analytes rapidly and on-site. Therefore, rapid, cheap, simple and on-site methods for the detection of analytes are greatly demanded [1].
In the last decades, because of the increasing and pressing demand for quick results, the scientific research has been focused more and more on the development and optimization of rapid methods of analysis. In particular, huge efforts have been made to develop portable, affordable and user-friendly systems for point-of-need testing (i.e., all the analyses performed directly where the sample is obtained), causing their exponential diffusion.
Immunochemical bioanalytical methods represent one of the most successful and versatile strategy for point-of-need applications. Among the different immunoassay-based tests, the Lateral Flow Assay (LFA), also known as immunochromatographic strip test technique, is increasing its relevance year-by-year [2] and has already found widespread applications in different fields [3]. The LFA technology is also very versatile as combines a number of variants such as test formats, recognition elements, signal reporters, and detection systems.
Thanks to its simplicity, rapidity, cost-effectiveness and no requirement of equipment or technical expertise, the LFA can be used in a decentralized or point-of-need testing strategy that requires little to no supporting infrastructure. Therefore, LFA is very useful especially in low-resource field environments and in developing Countries that cannot afford standard analytical instrumentation to perform analyses. Moreover, LFA can be the right ally of standard analytical methods in developed Countries, ensuring high sample throughput and rapid decision-making.
Historically, point-of-need tests have been considered to give less diagnostic accuracy than those used in centralized testing environments. However, there has been a terrific evolution in LFA technologies in the past decades and LFAs are becoming well suited to replace laboratory-based immunoassays in decentralized testing locations.
The relevant role of LFA technique can be also outlined through the optimism of the investors in this area and considering the global market size. In 2019 the global revenue market of LFAs is estimated in US$ 4,86 billion and it is projected to reach US$ 6,27 billion by 2024, growing at a compounded annual growth rate (CAGR) of 6.6 % [4].
Thanks to the advances of technologies, the LFA is being viewed more and more as a truly versatile technique, capable of more than adequate performance [5].
In this communication, several examples on the recent advances and some consideration on the future trends in LFA will be given.
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Biiooaannaalliittiiccaa22001199““FFrroommmmoolleeccuulleessttooddeevviicceess””-- 6 Dicembre 2019, Parma Gruppo Divisionale Bioanalitca, Divisione di Chimica Analitica, Società Chimica Italiana
References
[1] P. von Lode. Point-of-care immunotesting: approaching the analytical performance of central laboratory methods. Clin. Biochem. 2005, 38, 591-606.
[2] W. C. Mak, V. Beni, A. P. F. Turner. Lateral-flow technology: From visual to instrumental, TrAC. 2016, 79, 297-305.
[3] L. Anfossi, C. Baggiani, C. Giovannoli, G. D’Arco, G. Giraudi. Lateral-flow immunoassays for mycotoxins and phycotoxins: a review, Anal. Bioanal. Chem. 2013, 405, 467-480.
[4] https://www.marketstudyreport.com/reports/global-lateral-flow-assay-market-growth-status-and-outlook-2019-2024. Last accessed on 11.11.2019.
[5] I.Y. Goryacheva. Formats of Rapid Immunotests – Current-Day Formats, Perspectives, Pros and Cons. In: I.Y. Goryacheva (Eds), Rapid Immunotests for Clinical, Food and Environmental Applications. 1st ed., Elsevier B. V., 2016, 33-78.
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Biiooaannaalliittiiccaa22001199““FFrroommmmoolleeccuulleessttooddeevviicceess””-- 6 Dicembre 2019, Parma Gruppo Divisionale Bioanalitca, Divisione di Chimica Analitica, Società Chimica Italiana
Development of a new integrated analytical platform for the
identification of short peptides in cow milk
Carmela Maria Montonea, Sara Elsa Aitaa, Anna Laura Capriottia, Michela Antonellia, Andrea Cerratoa, Susy Piovesanaa, Aldo Laganàa,b
a Department of Chemistry, Università di Roma “La Sapienza”, Piazzale Aldo Moro 5, 00185 Rome, Italy b CNR NANOTEC, Campus Ecotekne, University of Salento, Via Monteroni, 73100 Lecce, Italy
e-mail: carmelamaria.montone@uniroma1.it
Short peptides are important compounds in a variety of fields, including food and nutraceutical applications, but also biomarker discovery, bioactive peptide discovery and peptide drug separation. Despite the importance of short peptides, they are currently less studied than other peptides because of the lack of dedicated methods for their characterization. In particular, in this work an untargeted peptidomic approach based on ultrahigh-performance liquid chromatography coupled to high-resolution mass spectrometry was developed for the identification of short peptides in cow milk samples. After milk defatting and precipitation, the sample was purified by cotton-hydrophilic interaction liquid chromatography (HILIC) micro tip in order to avoid suppression phenomena due to contaminants present in milk, such as carbohydrates. The sample was then separated by means of two chromatographic columns, with a complementary selectivity mechanism, namely reversed-phase C18 column and porous graphitic carbon (PGC). By this approach, the method allowed the separation and characterization of di-, tri- and tetrapeptides. A total of 57 and 41 peptides were identified by using a C18 and a PGC column, respectively; in particular, 31 were exclusively identified by using the C18 column, 15 unique peptides were identified by using the PGC column, while 26 were in common between the two data sets, demonstrating that the two columns have a different selectivity mechanism. The results indicated that an integrated approach may be appropriate to improve the separation of different peptides and increase the number of identifications because of the wide range of polarity of short peptides. The method allowed the untargeted identification of short peptides in milk, a complex matrix chosen as a representative real sample for method application, and provides complementary information to that accessible by ordinary peptidomics.[1]
Figure 1: Workflow analysis
References
[1] Carmela Maria Montone, Anna Laura Capriotti, Andrea Cerrato, Michela Antonelli, Giorgia La Barbera, Susy Piovesana, Aldo Laganà, Chiara Cavaliere. Identification of bioactive short peptides in cow milk by high-performance liquid chromatography on C18 and porous graphitic carbon coupled to high-resolution mass spectrometry. Analytical and Bioanalytical Chemistry. 2019, 411, 3395–3404
Bioanalitica 2019 “From molecules to devices” - 6 Dicembre 2019, Parma Gruppo Divisionale Bioanalitca, Divisione di Chimica Analitica, Società Chimica Italiana
Helical multi-walled carbon nanotubes coated fibers for
SPME determination of polycyclic aromatic hydrocarbons at
ultra-trace levels in ice cores
Angela Arcoleo, Federica Bianchi, Maria CareriDepartment of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, Parma (Italy)
e-mail: angela.arcoleo@unipr.it
Human impact on the Earth’s environment is recorded in the ice of the polar regions in the form of atmospheric bubbles and airborne particulate matter that are trapped in glaciers or ice caps during or shortly after snow deposition [1]. Persistent organic pollutants (POPs) such as Polycyclic Aromatic Hydrocarbons (PAHs) in ice cores are known to be clear markers of human activity [2]. The analysis of PAHs in ice core samples requires high sensitivity and selectivity due to the presence of these compounds at ultra-trace levels. In addition, the volume of sample available is normally of the order of tens of mL, depending on the temporal resolution required [1].
In a research program dealing with the development of selective solid-phase microextraction (SPME) coatings for the determination of environmental pollutants [3, 4], the aim of the present study was the development and validation of a SPME method coupled with gas chromatography- mass spectrometry (GC-MS) for the enrichment and determination of 16 US EPA priority pollutant PAHs in ice core samples. In order to improve the capabilities of this technique, helical multiwalled carbon nanotubes (HMWCNTs), a type of multiwalled carbon nanotubes (MWCNT) (Figure 1), were proposed as innovative SPME coatings. The potential of wide applications in separation science for carbon nanotubes is due to non-covalent forces including hydrogen bonding, electrostatic forces, stacking, hydrophobic interactions and van der Waals forces [5].
Figure 1: Structure of a MWCNT and b HMWCNT
The present method combined high sensitivity, ease of operation, simplicity, and environmental friendliness of SPME with high absorption capacity, resistance to organic solvents, thermal and chemical stability of MWCNTs [5]. Explicitly, HMWCNTs coated fibers proved to have high efficiency in adsorption and recovery for the 16 PAHs in water samples compared to different types of MWCNTs. Extraction conditions were optimized by investigating extraction time and extraction temperature as main factors applying a Central Composite Design; optimal conditions were identified by using the multicriteria method of the desirability functions. Method validation showed quantitation limits for the 16 PAHs ranging from 0.4 to 4.0 ng L-1 with RSD% within 28.4%
and recovery rates in the 93.70.1 – 119.70.2% range.
The good selectivity and the low volume consumption proved the potential application of the developed SPME-GC-MS method to snow and ice core samples for the detection of PAHs at ultra-trace levels.
a b
Bioanalitica 2019 “From molecules to devices” - 6 Dicembre 2019, Parma Gruppo Divisionale Bioanalitca, Divisione di Chimica Analitica, Società Chimica Italiana
References
[1] C. Barbante, A. Spolaor, W. R. Cairns, C. Boutron. Man’s footprint on the Arctic environment as revealed by analysis of ice and snow. Earth-Science Rev. 2017, 168, 218–231.
[2] J. Gabrieli, P. Vallelonga, G. Cozzi, P. Gabrielli, A. Gambaro, M. Sigl, F. Decet, M. Schwikowski, H. Gäggeler, C. Boutron, P. Cescon, C. Barbante. Post 17th-century changes of european PAH emissions recorded in high-altitude alpine snow and ice. Environ. Sci. Technol. 2010, 44, 3260–3266.
[3] F. Bianchi, F. Bisceglie, M. Careri, S. Di Berardino, A. Mangia, M. Musci. Innovative sol-gel coatings for solid-phase microextraction. Development of fibers for the determination of polycyclic aromatic hydrocarbons at trace level in water. J. Chromatogr. A. 2008, 1196–1197, 15–22.
[4] N. Riboni, J.W. Trzcinski, F. Bianchi, C. Massera, R. Pinalli, L. Sidisky, E. Dalcanale, M. Careri. Conformationally blocked quinoxaline cavitand as solid-phase microextraction coating for the selective detection of BTEX in air. Anal Chim Acta. 2016, 905, 79–84.
Exploring the undisclosed hemp compounds: the
comprehensive polar lipidome tool for the enrichment,
fractionation and identification
Michela Antonellia, Sara Elsa Aitaa, Barbara Benedettia, Chiara Cavalierea, Andrea
Cerratoa, Aldo Laganàa,b
aDepartment of Chemistry, University of Rome “La Sapienza”, Piazzale Aldo Moro 5,
00185 Rome, Italy
bCNR NANOTEC, Campus Ecotekne, University of Salento, Via Monteroni, 73100 Lecce,
Italy
e-mail: michela.antonelli@uniroma1.it
Cannabis Sativa L. has been well known for its multiple purposes for medical, recreational
and textile application [1,2]. Cannabis chemical composition is characterized by more than 400 chemical entities and cannabinoids represent more than 15% of the total. Along with cannabinoids, several other constituents were detected and characterized in hemp. For example, the terpenes, responsibles for the odor and flavor of the different cannabis strains [3], were used as markers to establish the chemotaxonomy of the plant, along with cannabinoids [4]. Moreover, Cannabis fruits and roots contain phenolic compounds, including, for instance, cannflavin A and B, which exert a wide range of biological activities [5]. Phytocannabinoids, terpenes, phenolic compounds and other minor components, such as fatty acids, amino acids, proteins, sugars, vitamins and pigments, provide significant human benefits, due to both synergic and antagonist interactions [6].Nevertheless, a detailed lipidome characterization is completely lacking in the literature. To achieve this goal, an extraction and enrichment procedure were developed for the characterization of phospholipids and sulfolipids. Firstly, a study on the solid-liquid extraction was performed, to maximize the recovery of the considered lipids; the best procedure consisted in a simple extraction with a mixture of methanol and chloroform (1:1, v/v). The hemp extracts were analyzed by ultra-high performance liquid chromatography coupled to high resolution mass spectrometry and lipids were tentatively identified by Lipostar. To improve the number of identifications, an enrichment method, based on graphitized carbon black solid phase extraction, was evaluated to fractionate phospholipids and sulfolipids into separate eluates. Recovery and matrix effect of the procedure were determined on a mixture of standard lipids, containing representative compounds for each considered lipid class. The optimized method allowed the tentative identification of 189 lipids, including 52 phospholipids and 80 sulfolipids, in the first and second fractions, respectively. The detection of only 6 sulfolipids in the first fraction and 9 phospholipids in the second fraction proved the efficacy of the fractionation method, which also allowed to increase the number of lipid identifications compared to the same procedure without enrichment, which scored 100 lipids. Finally, a semi-quantitative analysis permitted to characterize the hemp polar lipidome (Fig. 1). The results of this study allow to improve the knowledge on hemp chemical composition with a detailed description of its phospho- and sulfolipid profiles.
Figure 1: Relative abundances (%) of the identified lipid species in the first GCB-SPE eluate (a) and second
eluate (b) reported according to main lipid classes. PL indicates phospholipids.
References
[1] Russo EB, Jiang HE, Li X, Sutton A, Carboni A, Del Bianco F, Mandolino G, Potter DJ, Zhao YX, Bera S, Zhang YB, Lü EG, Ferguson DK, Hueber F, Zhao LC, Liu CJ, Wang YF, Li CS. Phytochemical and genetic analyses of ancient cannabis from Central Asia. J Exp Bot. 2008; 59(15):4171–82.
[2] Skoglund G, Nockert M, Holst B. Viking and early middle ages northern scandinavian textiles proven to be made with hemp. Sci Rep. 2013; 3:1–6.
[3] Flores-Sanchez IJ, Verpoorte R. Secondary metabolism in cannabis. Phytochem Rev. 2008; 7(3):615–39. [4] Elzinga S, Fischedick J, Podkolinski R, Raber JC. Cannabinoids and terpenes as chemotaxonomic markers in Cannabis. Nat Prod Chem Res. 2015;03(04):1–9.
[5] Atakan Z. Cannabis, a complex plant: different compounds and different effects on individuals. Ther Adv Psychopharmacol. 2012; 2(6):241–54.
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Biiooaannaalliittiiccaa22001199““FFrroommmmoolleeccuulleessttooddeevviicceess””-- 6 Dicembre 2019, Parma Gruppo Divisionale Bioanalitca, Divisione di Chimica Analitica, Società Chimica Italiana
Drug development and loading study of polypeptide-drug
conjugates through asymmetrical flow field-flow fractionation -
multidetection
Valentina Marassia, b, Juan J. Arroyoc, María J. Vicentc, Barbara Rodaa, b, Pierluigi Reschigliana,b, Andrea Zattonia,b
a Department of Chemistry “G. Ciamician”, University of Bologna, Bologna, (Italy) b byFlow Srl, Bologna, Italy
c Polymer Therapeutics Lab. Príncipe Felipe Research Institute, Valencia (Spain) email: valentina.marassi2@unibo.it
Among the most promising polymeric therapeutic scaffolds proposed for drug delivery, poly-glutamic acid (PGA) polypeptides can be advantageous given their biodegradability on the main chain, furthermore allowing for the use of higher molecular weight polymers for pharmacokinetic optimization [1].
Covalently conjugated drugs or imaging agents on the polypeptide can introduce new spatial and electrostatic contributions that modify drastically the whole molecule equilibrium, impacting size, conformation and biophysical properties.
PGA scaffolds are made of a succession of glutamic monomers linked trough peptidic bonds, and are soluble in water and physiological buffers: however, hydrophobic bioactive or imaging moieties attribute unique properties in the nanoscale, promoting different structural and conformational effects influencing solubility, behaviour and biological performance [2].
With the goal of studying new based therapeutic polypeptides we analysed different PGA-Doxorubicin (Dox) conjugates by asymmetrical flow field-flow fractionation (AF4) followed by multidetection. This analytical platform, allowing native in-flow characterisation, is able to provide a data set (RMS radius, fluorescence and absorption profile, retention behaviour) which can be easily correlated to in-vitro applicability [3].
With an increasing loading of Dox on the PGA polymer (through hydrazone link), we observed a slight increase in retention time (hence a higher hydrodynamic radius), compatible with the increased total mass of the conjugated systems, but more interestingly a strong decrease in RMS radius, indicating the rearrangement of the conjugated structures towards a more compact state.
An insight of the mechanism driving this structure change can be obtained by the Fluorescence spectra tuned on Dox absorption/emission. In this context, the same amount of Dox was kept constant in AF4 injections. Indeed, Dox-specific fluorescence intensity changes with loading, but the highest fluorescence was induced by the lowest-loaded conjugate, suggesting that the drug localization is not constant.
Likely, the conjugation of small amounts of Dox promotes a coronal disposition of the drug and its intermolecular repulsions could support an opened and larger structure. With an increased loading, π-stacking interactions could become prevalent and the structure could start compacting in smaller and denser disposition, with Dox organized in a central hydrophobic core. A clear trend to decrease in RMS values when the loading of Dox increased up to 26% wt was identified for several PGA-hyd-Dox single conjugates.
The biological implications of this trend in terms of cellular uptake were investigated: however Doxorubicine was subject to strong interference and loss of linearity in the signal from the cytometer, possibly also due to the changing drug localization in the carrier.
A new family of PGA-Dox conjugates bearing a similar amount of Cyan also conjugated to the same polymeric backbone was then synthesized. Following this approach, the perspective is to be able to track the fluorescent dye inside the cells rather than the drug. On the other hand it
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Biiooaannaalliittiiccaa22001199““FFrroommmmoolleeccuulleessttooddeevviicceess””-- 6 Dicembre 2019, Parma Gruppo Divisionale Bioanalitca, Divisione di Chimica Analitica, Società Chimica Italiana
was necessary to assess whether, 1. Despite having incorporated the blue dye moiety, the nanoconstruct preserved the already observed behavior and 2. The drug loading kept driving the RMS shift in solution.
Both hypotheses proved to be true, though the highest drug loading was less soluble, had a very low signal at the conjugate retention time (with a higher RMS radius measured), and a higher signal at later times, compatible with aggregates.
Figure 1: loading-dependent size changes in PGA-Cyan-Dox conjugates
The results indicate that high-Dox loading could collapse the system possibly even re-extruding part of the drug on the particles surface, while also causing precipitation. The highest loading also displayed the lowest recovery (36% against >90%) supporting the change in surface chemistry and consequent interaction with the AF4 membrane.
This hypothesis justifies confusing in-vitro information suggesting that there is a “critical” amount of loadable drug able to retain the carrier structure, and that above that value delivery and biodistribution are hindered together with solubility. This also impacts Cyan fluorescence that is quenched at the highest Dox loading.
The characterization approach presented can therefore be fundamental in pre-screening drug-polymer therapeutic agents with different drug and/or dye combinations, and in understanding how to develop the highest-loaded functional structures, thus reducing in-vitro and in-vivo screenings of underperforming candidates.
References
[1] A. Duro-Castano, I. Conejos-Sanchez, M.J. Vicent. Peptide-Based Polymer Therapeutics. Polymers 2014, 6 (2), 515-551
[2] O. Zagorodko, J.J. Arroyo-Crespo, V.J. Nebot, M.J. Vicent. Polypeptide‐Based Conjugates as Therapeutics: Opportunities and Challenges. Macromolecular Bioscience 2017, 17 (1), 22
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Biiooaannaalliittiiccaa22001199““FFrroommmmoolleeccuulleessttooddeevviicceess””-- 6 Dicembre 2019, Parma Gruppo Divisionale Bioanalitca, Divisione di Chimica Analitica, Società Chimica Italiana
3D printed alginate/nanocrystalline cellulose-based scaffolds
loaded with silver nanoparticles combining antimicrobial and
cytotoxic effects towards HepG2 cell line
aCarlo Bergonzi, aSilvia Marando, bLaura Bergamonti, bClaudia Graiff, cMaria Cristina Ossiprandi, aIlaria Zanotti, aFranco Bernini, aRuggero Bettini, aLisa Elviri.
aFood and Drug Department, University of Parma, V.le delle Scienze 17/a, 43124, PR.
bDepartment of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, V.le delle Scienze
27/A, 43124, Parma.
cDepartment of Veterinary Science, University of Parma, Strada del Taglio 10, 43126, Parma, Italy.
e-mail: lisa.elviri@unipr.it
The growing demand for personalized medicine requires innovation in drug manufacturing to combine versatility with automation and cost effectiveness. Here, innovative freeze-deposition 3D printing is explored for the production of alginate (ALG)/nanocrystalline cellulose (CNC)-based hydrogel scaffolds loaded with silver nanoparticles (AgNP’s) (Figure 1, a).
ALG as well as CNC are biocompatible materials extremely employed in tissue engineering for implant scaffolding for tissue regeneration and cure. Adequate mechanical properties, microstructural three-dimensionality, antimicrobial activity towards microbial pathogens and cytotoxicity versus tumor cells are crucial requirements for advanced devices targeting such application[3].
CNC was dispersed in a 100 ppm AgNP’s suspension, once achieved homogenization under magnetic stirring, alginate was added in little amounts till the final concentration of 5% w/v. The formulation was exploited for scaffold production through an in-house-built 3D printer machine, subsequent fixation of the three-dimensional structure occurred by gelation in a 270 mM CaCl2
solution [1]. HPLC SEC-UV/VIS analysis was performed to compare stock NP’s suspension and scaffolds in terms of AgNP’s size while Dynamic Light Scattering (DLS) was used to characterize NP’s size distribution; furthermore, Scanning Electron Microscopy (SEM) analysis were crucial for determination of scaffold morphology/topography and NP’s conformation. CNC is widely known for its capacity to strengthen mechanical properties of materials dedicated to bioengineering [2], tensile traction tests were thus conducted through a dynamometer and Young’s modulus (Ym) of the
hydrogel scaffolds was calculated in order to investigate physical suitability of the system with the potential host tissue. Water content was measured by gravimetric analysis to have a more complete scenario on the chemico-physical features of the object. Since fight again recurrent infections is a major challenge in implant application, antimicrobial activity of the scaffolds was assayed by Mueller Hinton agar diffusion method [5]; bacterial inocula of S. aureus and P. aeruginosa were plated on Petri dishes and circular 6 mm diameter scaffolds were applied on the terrain surfaces, finally, growth inhibition area around samples was measured after 24, 48 h. Antitumor property was instead assessed seeding HepG2 cells on scaffolds, monitoring their viability by microscopy and MTT assays over 8 days and performing shotgun targeted liquid chromatography-mass spectrometry (LC-MS) for the quantification of pro-proliferative and apoptotic proteins.
HPLC SEC-UV/VIS chromatograms were compared showing no significant differences, meaning NP’s distribution did not change over the whole process. It is reported that nanoparticle size influences NP’s antibacterial effectiveness, DLS analysis indicated a mean AgNP’s hydrodynamic diameter of 60.29 nm and a Polydispersity Index of 0.377, suggesting a sufficient antimicrobial efficacy in accordance with Martinez-Castanon et al. (2008) [6]; confirmed by the fact that all scaffolds showed promising in vitro antimicrobial activity (100% of growth inhibition) against S. aureus and P. aeruginosa (Figure 1; e, f, respectively). SEM identified AgNP’s resulted spherical and their diameter was confirmed with respect to DLS results (Figure 1; c, d), furthermore, macro/micro morphologies established in the design phase were respected [1] showing a three-dimensional highly porous structure (Figure 1; b). Ym resulted 0.54 ± 0.03 Mpa , that indicate the
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Biiooaannaalliittiiccaa22001199““FFrroommmmoolleeccuulleessttooddeevviicceess””-- 6 Dicembre 2019, Parma Gruppo Divisionale Bioanalitca, Divisione di Chimica Analitica, Società Chimica Italiana
retained great amounts of water: 87.12 ± 0.64%, moisture is considered among biocompatibility indexes for re-growing healing tissue since it permits cell survival and their physiologic metabolism. Three-dimensionality of the printed objects was maintained over all the production process and subsequent conservation; once started in vitro tests, scaffolds without CNC (only calcium alginate hydrogel) lost their 3D structure in 2 days when HepG2 cells grew on them, triggering fast release of NP’s and showing an acute cytotoxic effect (vitality reduced till 8 ± 1% at day 2). On the contrary, the presence of CNC allowed the physical maintenance of the 3D structure and cytotoxic effect towards tumor cells over time, probably due to sustained NP’s release from the stiffer biomaterial (vitality reduced till 1.5 ± 0.5 % at day 8).
Shotgun targeted LC-MS proteomic analysis revealed that relative abundancies of proliferative markers, particularly Ki-67, were significantly higher in AgNP’s-free conditions, whereas those concerning apoptotic proteins, such as BAX and caspases, resulted much higher in samples treated with AgNP’s.
Scaffolds were analytically characterized showing chemico-physical parameters that resulted suitable for implant applications. Moreover, it was observed that the alginate system developed avoided the aggregation of AgNP’s, in addition, their controlled release can be modulated by selecting ALG or ALG-CNC combined biomaterials providing a proof of concept on the versatility of the process.
References
[1] Elviri et al. Highly defined 3D printed chitosan scaffolds featuring improved cell growth. Biomed Mater 2017, 12. [2] McKee et al. Thermoresponsive Nanocellulose Hydrogels with Tunable Mechanical Properties. ACS Macro Lett. 2014, 3, 266-270.
[3] Rees et al. 3D Bioprinting of Carboxymethylated-Periodate Oxidized Nanocellulose Constructs for Wound Dressing Applications. BioMed Research International Vol 2015, Article ID 925757, 7 pages.
[4] Tirella et al. PAM2 (Piston Assisted Microsyringe): A New Rapid Prototyping Technique for Biofabrication of Cell Incorporated Scaffolds. TISSUE ENGINEERING: Part C 2011, 17, No 2.
[5] Heatley. A method for the assay of penicillin. Biochem J. 1944, 38, 61-65.
A PAPER-BASED ELECTROCHEMICAL DEVICE FOR THE
DETECTION OF PESTICIDES INSPIRED BY NATURE: A
FLOWER-LIKE ORIGAMI BIOSENSOR
Fabiana Arduini1, Veronica Caratelli1, Greta Fegatelli1, Giorgia Ciampaglia1, G. Palleschi1, D. Moscone1
1Department of Chemical Science and Technologies, University of Rome Tor Vergata, Via della Ricerca Scientifica, 00133 Rome, Italy
e-mail: fabiana.arduini@uniroma2.it
Pesticides are largely used at worldwide level to improve the food production, fulfilling the needs of the global population, which is increasing year by year. Although persistent pesticides (e.g. DDT) have been replaced with less persistent ones, contamination of food, soil and water by pesticides remains an issue of public concern. To better manage this problem, EU sets regulations for a sustainable employment of pesticides by promoting the adoption of Integrated Pest Management (Directive 2009/128/EC). In this overall scenario, the detection of pesticides in liquid and aerosol solutions at low concentrations (ppb level) is required to accomplish the regulatory aspect and to preserve the health of environment and human being. Herein, we propose a flower-like origami paper-based device for the detection of several classes of pesticides by combining different enzyme-inhibition biosensors. This device was developed by integrating two different office paper-based screen-printed electrodes and multiple filter paper-based pads to load enzymes and enzymatic substrates. The versatile analysis of different pesticides was carried out by folding and unfolding the filter paper-based structure, without any addition of reagents and any sample treatment (i.e. dilution, filtration, pH adjustment) [1]. The paper-based platform was employed to detect paraoxon, 2,4-dichlorophenoxyacetic acid, atrazine and glyphosate at ppb level by exploiting the capability of these different types of pesticides to inhibit butyrylcholinesterase, alkaline phosphatase, tyrosinase, and peroxidase enzymes respectively. The degree of inhibition, correlated to the quantity of pesticides, was chronoamperometrically evaluated, monitoring the enzymatic activity in the absence and in the presence of pesticides by using a portable potentiostat. To improve the sensitivity, the paper-based electrodes were modified with carbon black nanoparticles in the case of platforms for 2,4-dichlorophenoxyacetic acid, atrazine and glyphosate detection or carbon black decorated with Prussian blue nanoparticles for the detection of paraoxon. Preliminary results for the detection of 2,4 D herbicide in gas phase were obtained using a commercial portable nebuliser (SIMBR aerosol system). The corresponding calibration curve is characterised by the following equation y = (7 ± 2) + (0.110 ± 0.009) x with a R2 = 0.937 and a LOD (calculated as the amount of drug which gives 10% of inhibition) equal to 30 ppb.
References
[1] F. Arduini, S. Cinti, V. Caratelli, L. Amendola, G. Palleschi, D. Moscone. Origami multiple paper-based electrochemical biosensors for pesticide detection. Biosens. Bioelectron. 2019, 126, 346-354.
A rapid and sensitive lateral flow immunoassay for the real
time monitoring of the compliance in antiretroviral therapies:
from hapten synthesis to prototype.
Cavalera S.1, Di Nardo F.1, Anfossi L.1, Baggiani C.1, Simiele M.2, D'Avolio A.2, Mercader J.V.3, Abad-Fuentes A.3, Agullό C.4 and Abad-Somovilla A.4.
1University of Torino, Department of Chemistry, Torino, Italy
2CoQuaLab, University of Torino, Department of Medical Sciences, Torino,Italy 3CSIC, Instituto de Agroquímica y Tecnologia de Aliméntos, Paterna (Valencia), Spain
4Universitat de Valencia, Facultat de Química Organica, Paterna (Valencia), Spain e-mail: simone.cavalera@unito.it
The High Activity Anti-Retroviral Treatment (HAART) approach, which combines antiretroviral drugs in fixed-dose administrations, is still a milestone in the HIV infection management, facilitating better clinical performances and a decrease in the risk of contagion and consequent probability to decline towards the Acquired Immuno-Deficiency Syndrome (AIDS) condition1,2. However, a low adherence to the therapy still holds as the main source of therapeutic failure, hence the need of efficient tools to monitor the compliance3. The real-time monitoring of ARV drugs is usually made by using expensive, time consuming and poorly accessible instrumental methods that demonstrate an overall incompatibility despite of the excellent analytical performances4. The efficient and versatile technology of immunochromatographic strip test (ICST) could fulfill many requirements, such as low cost devices, rapid analyses, reliability and availability. The main aim of this study is the development of an ICST, or lateral flow device, to semi-quantitatively determine an adherence-related amount of drug in HIV positive patient's saliva samples5.The target ARV molecule was chosen among drugs currently present in several fixed-dose combination and considered as "surviving" drugs in future formulations. Because of the absence of available bioreagents for the development of the LFIA (such as anti-ARVdrug antibodies and ARVdrug derivatives), ab initio organic synthesis was successfully applied to prepare the immunogenic ARV drug derivative and the capturing ARV drug derivative. The immunogen has been inoculated in 3 mice, from which 6 monoclonal antibodies (mAbs) were obtained. The mAbs were characterized by enzyme-linked immunosorbent assay (ELISA) and 3 clones were selected basing on their affinity and selectivity towards the target molecule. The mAbs employed to develop a lateral flow immunoassay showed high sensitivity (e.g. mAb 321, IC50 = 0,12nM) and applicability to
the detection of the ARV in human urine and saliva. The developed LFIA device allows for assessing the adherence to HAART and do not require equipment, nor trained operators .These outcomes represent a breakthrough on the current state-of-the-art of therapeutic drug monitoring and can draw a new path towards the definitive management of HIV positive patients.
Figure 1: ELISA characterization References
1. Günthard, H. F. et al. Antiretroviral drugs for treatment and prevention of HIV infection in Adults: 2016 recommendations of the international antiviral society
316, 191–210 (2016).
2. Bezabhe, W. M., Chalmers, L., Bereznicki, L. R. & Peterson, G. M. Adherence to Antiretroviral Therapy and Virologic Failure.
3. El-Zailik, A., Cheung, L. K., Wang, Y., Sherman, V. & Chow, D. S. L. Simultaneous LC
of simvastatin, atorvastatin, rosuvastatin and their active metabolites for plasma samples of obese patients underwent gastric bypass surgery.
doi:10.1016/j.jpba.2018.10.045
4. Zhang, Y. & Zhang, R. Recent advances in anal immunosuppressive drugs. Drug Test. Anal.
5. Anfossi, L., Di Nardo, F., Cavalera, S., Giovannoli, C. & immunoassay: An overview of strategies towards high 9, (2018).
ARV drug concentration (nM)
Ab so rb an ce (a .u .)
ELISA characterization (left) and developed ICST device testing one mAbs for detecting the ARVdrug.
Antiretroviral drugs for treatment and prevention of HIV infection in Adults: 2016 recommendations of the international antiviral society-USA Panel. JAMA
Bezabhe, W. M., Chalmers, L., Bereznicki, L. R. & Peterson, G. M. Adherence to Antiretroviral Therapy and Virologic Failure. 95, 1–9 (2016).
Zailik, A., Cheung, L. K., Wang, Y., Sherman, V. & Chow, D. S. L. Simultaneous LC
of simvastatin, atorvastatin, rosuvastatin and their active metabolites for plasma samples of obese patients underwent gastric bypass surgery. J. Pharm. Biomed. Anal.
doi:10.1016/j.jpba.2018.10.045
Zhang, Y. & Zhang, R. Recent advances in analytical methods for the therapeutic drug monitoring of Drug Test. Anal. 10, 81–94 (2018).
Anfossi, L., Di Nardo, F., Cavalera, S., Giovannoli, C. & Baggiani, C. Multiplex lateral flow immunoassay: An overview of strategies towards high-throughput point-of-need testing.
9,00 1,80 0,36 0,07 0,01
-) ARV drug concentration (nM
Ab so rb an ce (a .u .)
one mAbs for detecting the
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Biiooaannaalliittiiccaa22001199““FFrroommmmoolleeccuulleessttooddeevviicceess””-- 6 Dicembre 2019, Parma Gruppo Divisionale Bioanalitca, Divisione di Chimica Analitica, Società Chimica Italiana
ORIGAMI PAPER-BASED SENSORS AND PRECISION MEDICINE:
AN APPLICATION IN ALZHEIMER DISEASE
Veronica Caratellia, Adriana Ciampagliaa, Jacopo Guiduccia, Danila Mosconea, Fabiana Arduinia
a Department of Chemical Science and Technologies, University of Rome “Tor Vergata”, Rome (Italy)
e-mail: veronica.caratelli@uniroma2.it
The last decades have seen an increase of higher mortality rates and healthcare costs for treatment, hospitalization and care assistance for Alzheimer and Parkinson diseases. To this regard, the cost for managing patients affected by neurodegenerative disorders has been estimated to be approximately € 130 billion/year [1]. The recent trends of precision medicine have boosted the personalization of medical care in several field including neurological diseases, thanks to new diagnostics and therapeutics developed and discovered, respectively [2]. In this overall scenario, we propose a novel paper-based lab-on-a-chip to deliver a cost-effective and easy to use sensing tool for a customised administration of drugs for Alzheimer disease. Among several drugs, we have designed the device for evaluating the efficiency of alkaloid compounds (e.g. Physostigmine) which can inhibit in a reversible way the cholinesterase enzyme. Since the activity of cholinesterase is different among the patients, the administration of the customised amount of drug can improve the treatment and the quality of patient life, avoiding side effects due to the overdosage. In detail, we exploited office paper to print the electrode and Vivid™ Plasma Separation membrane to threat the blood sample as well as to load the reagents needed for the measurement, delivering a reagent free analytical tool. The calibration curve of BChE obtained in real blood sample gave a linearity between 2 and 12 U/mL with a sensitivity of 0.050 ± 0.004 µA mL/U. The Physostigmine inhibition activity against cholinesterase enzyme was measured in matrix in the range between 0.01 and 1 µM and a linearity up to 0.5 µM was obtained, characterised by the following equation y = (9 ± 2) + (111.4 ± 7.1) x with a R2 = 0.960 and a LOD (calculated as the amount of drug which gives 10% of
inhibition) = 0.009 µM.
References
[1] C. Strafella et al. Application of Precision Medicine in Neurodegenerative Diseases. Frontiers in neurology,
2018, 9, 701.
[2] L.Tan, T. Jiang, L. Tan, L.J.T. Yu. Toward precision medicine in neurological diseases. Ann Transl Med, 2016, 4, 104.
