minunni affinity biosensors

AffinityBiosensors MariaMinunni DipartimentodiChimica,PoloScientifico, Università degliStudidiFirenze, ViadellaLastruccia3,50019SestoF.no(FI) Italy ChemicalSensor A device responding in a fast and reversible fashion to chemical stimuli, i.e. changes in concentration of a biochemicalspecies M.Varcacel,Quim.Anal.,9(1990)215 Abiosensorisanwholeandintegrateddeviceprovidinganalyticalinformation(qualitativeor semi/quantitative) by using a biomolecular recognition element (biochemical receptor) in closespatialcontactwithatransducer. Thetransducerconvertsthechemicaleventintoarecordablesignal. The Biosensor Twoclassesofbiosensors: Enzymatic(catalytic) Thebiologicalelement (enzyme)convertsthe substrate intoa product SP Thetransducerreveals S orP Affinity Thebiologicalelement (receptor)binds specificallytheanalyte leadingtoa complex A +BAB Thetransducerreveals the complex BIOSENSORSGENERAL Minimal sample pretratment Userdefinedtarget specificitydictatedby choiceofimmobilised ligand. Affinity&kineticmeasurements Qualitativeranking; Calculationofobservedaffinity constants;Calculationofkinetic constants. Lowreagentconsumption Miniaturization. Highsamplethroughput Autosampling Flowsystems On‐lineconfiguration Rapidanalysis Equilibrium usuallynotnecessary Dataacquisition,storage& processingUsuallyfacilitatedby dedicatedsoftware. Non‐invasivelabel‐freetechnology Eliminatestimeconsumingandcostly labelingprocedures. Minimaldemandonusertimeorskills Automatedanalysis Requirements OPTIMAL BIOSENSOR performance BIOSENSORSGENERAL Thefollowinglistdescribessomeofthecurrentapplications. • Clinicaldiagnosisandbiomedicine • Farm,gardenandveterinaryanalysis • Processcontrol:fermentationcontrolandanalysis • Foodanddrinkproductionandanalysis • Microbiology:bacterialandviralanalysis • Pharmaceuticalanddruganalysis • Industrialeffluentcontrol • Pollutioncontrolandmonitoring • Mining,industrialandtoxicgases • Militaryapplications Where are Biosensors Being Used? Glucosestillaccountsfor87%ofthemarket WorldMarketin2007=US$9.4billion 0 1 2 3 4 5 6 7 8 9 10 1996 1999 2001 2003 2005 2007 US$b CourtesyofProf.A.P.F.Turner,CranfieldUniversity,UK BIOSENSORSGENERAL Load lancet into launcher and reassemble launcher Load lancet into launcher and reassemble launcher Prick finger or arm Prick finger or arm Deposit blood drop on to test strip & insert strip Deposit blood drop on to test strip & insert strip Read test strip Read test strip Dispose of materials Dispose of materials 1-2 Minutes COMMERCIALBIOSENSORS:DISPOSABLEandPORTABLE USERFRIENDLY,FASTANALYSISINCOMPLEXMATRICES ParadigmaticexampleofCatalyticBiosensor: BloodGlucoseMonitoring COMMERCIALBIOSENSORS:MULTI‐USE,BENCHINSTRUMENTATION ParadigmaticexampleofAffinity‐BasedBiosensors(ABBs): 1990BIACORE™ SurfacePlasmonResonance(SPR) StudyAffinityreactionsonAusurfaces Salesabout100peryear Opticallabel‐freesensing Early’90s‐present •Immunosensors, SensorsbasedonAntibody‐Antigen interaction:Immunocomplexformation ANTIBODY(immunoglobulin):Abiologicalmolecule(protein)thatspecificallyrecognizesaforeign substance(antigen)asameansofnaturaldefence UseofAntibody inanalyticalchemistry: Immunoassays(IAs)ELISA,RIA,aretechniquesusedinclinicalchemistry, beingusedforthefastandsafedetectionofproteins,hormones,and pharmaceuticalagents Late’90s‐present: Nucleicacid‐basedsensors • Detectionofsequencesbyhybridizationreactionandcomplexformation (dsDNA) TargetDNAsequences(i.e.GMOs,bovinesatelliteDNA,bacteria etc.): food control , • Detectionoftargetanalyteswithaffinityinteractionmakinguseofnew receptors,calledAptamers,producedinvitro(insteadofAbs):Aptasensors TargetProteins(i.e.TAT,trombin)bytheirabilitytointeractwithAptamers (nucleicacidsselectedinvitro)):diagnostic Approachesin(bio)sensingas analyticalchemists • Opticalsensing • Piezoelectricsensing • Electrochemicalsensing MariaMinunni Researcher IlariaPalchetti Researcher GiovannaMarrazza AssociateProfessor MarcoMascini FullProfessorinAnalyticalChemsitry Staff:4permanent [...]... i.e. glucose in blood (glicemia), phenols in food and environment: enzymatic‐sensing • protein (i.e antigen or antibody detection): affinity immunosensing or aptasensing; • target  DNA sequences (bacteria, viruses, transgenes –GMOs or  gene doping) affinity DNA sensing; • Point  mutations:  beta globin gene, p53 gene • Drug ‐ receptor interaction i.e.  alcaloids with DNA BIOSENSORS:  CASE STUDIES Affinity sensing for: Development of a nucleic acids‐based sensors:  ... Promoter P35 in patented GM food = marker sequence GMOs: STRATEGIES BIOSENSORS Optical (SPR) CRM, real samples P35S, Tnos, G Feriotto, , J Agric Food Chem 50 (2002) 955 E Mariotti, M Minunni, M Mascini, Analytica Chimica Acta, 453 (2002) 165 E Giakoumaki, et al., Biosensor and Bioelectronics, 19 (2003) 337 R Wang, et al., , Biosensors and Bioelectronics, 20 (2004) 598 M, Minunni, Spectroscopy, 17 (2003) 613 R Wang, et... SPR IMAGING (SPRI) FOR BIOSENSING: AN INNOVATIVE LABEL‐FREE, MULTIARRAY PLATFORM FOR THE  DETECTION OF BIOAFFINITY INTERACTIONS Surface  Plasmon Resonance (SPR) RU Array of Light  Sensitive Diodes  (LSD)  Sensorgram Biochip Time Sensing Area SPR imaging (SPRi) Sensorgrams Charge Coupled  Device (CCD)  ΔR% Time Biochip Sensing Areas: Regions of Interest (ROIs) S. Scarano, A.P. Turner, M. Mascini and M. Minunni,  Surface Plasmon Resonance Imaging For Affinity Based Biosensors,  in press on Biosens. ... inhibition sensors), pathogens Biosensors 2008 Word Conference Themes 2008 themes Nanomaterials & nanoanalytical systems Bioelectronics & biofuel cells Systems integration, proteomics and single cell analysis DNA chips & nucleic acid sensors Organism- and w hole cell-based biosensors New signal transduction technology Commercial developments, manufacturing and markets Enzyme-based biosensors Natural & synthetic receptors for biosensors. .. and papayas P35S, J Liu et al., Biosensors and Bioelectronics 20 (2004) 436 Electrochemical immunosensor soybean Cry protein Microchip CE CRM P35S, TNOS P Bogani et Food Chem, 113, 658-664, 2009 B Meric, et al., Food Control 15 (2004) 621 Volpe, G al., Analytical Letters (2006), 39(8), 1599-1609 J P Obeid., et al., Electrophoresis, 25 (2004) 922 Reference person:  Maria Minunni,  Marco Mascini  Optical sensing... 3 Immobilization chemistry • Point 1 ‐defintion of analytical problem and point 2 ‐biological  receptors Development of analytical protocol  for real application of sensors  and biosensors:   coupling transducer and suitable receptors  (affinity or catalytic based sensing) • Point 3: immobilization chemistries Development of immobilization chemistries suitable for the  different  sensing surfaces (i.e gold, graphite, etc... Vector cassette marker promoter gene Reflectivity sample 0.45 0.40 0.35 0.30 ΔR 0.2 5 0.20 0.00 0 5 10 15 20 Time (min.) An Integrated Approach based on  Affinity based Biosensing (ABBs) for  Gene Doping detection:  A Pilot Study Coordinator: M. Minunni Partners  UNIFI • Prof. Annarosa Arcangeli, Dipartimento di Patologia e Oncologia Sperimentali • Prof. Marcello Buiatti, Dip. di Biologia Evoluzionistica “Leo Pardi”... Biosensors and Bioelectronics, 20 (2004) 598 M, Minunni, Spectroscopy, 17 (2003) 613 R Wang, et al., , Biosensor and Bioelectronics, 20 (2004) 967 QCM CRM, real samples P35S, Tnos, M Minunni et al, Analytical Letters; 34 (2001) 825 M Minunni et al., Fresenius' Journal of Analytical Chemistry, 369 (2001) 589 I Mannelli et al., Biosensor and Bioelectronics, 18 (2003) 129 QCM CRM, food chain P35S, Electrochemical... 2001‐2004  EU (FP5)  GAPOGROWTH, Growth of Large GaPO4 Single Crystals and their  2002‐2005  EU (FP5)  use for Special Sensor Applications  Evaluation/validation of novel biosensors in real environmental  and food samples (VALIDATION OF BIOSENSORS)   2000‐2003  EU (FP5)  Sensors for monitoring water pollution from contaminated land,  1998‐2002  EU (FP5)  landfills and sediments Reference number  NMP4‐CT‐2006‐... Reference person: Ilaria Palchetti, Giovanna Marrazza,  Marco Mascini  Scanning electrochemical microscope (SECM) Electrochemical Multi‐arrays  Immuspeed diagnoSwiss S.A.‐Switzerland GRAVI™‐ Chip:  Biosensor for ultra‐ fast ELISA Where are we now? Biosensors are already established for: Health • For home or field use by non‐specialists e.g. home blood glucose  monitoring  • Small enough to be implanted in the body e.g. Implantable sensors  for glucose . Affinity Biosensors Maria Minunni DipartimentodiChimica,PoloScientifico, Università degliStudidiFirenze, ViadellaLastruccia3,50019SestoF.no(FI) Italy ChemicalSensor A . BloodGlucoseMonitoring COMMERCIAL BIOSENSORS: MULTI‐USE,BENCHINSTRUMENTATION Paradigmaticexampleof Affinity Based Biosensors (ABBs): 1990BIACORE™ SurfacePlasmonResonance(SPR) Study Affinity reactionsonAusurfaces Salesabout100peryear Opticallabel‐freesensing Early’90s‐present •Immunosensors, SensorsbasedonAntibody‐Antigen . , Biosensors and Bioelectronics, 20 (2004) 598 M, Minunni, Spectroscopy, 17 (2003) 613 R. Wang, et al., , Biosensor and Bioelectronics, 20 (2004) 967 QCM CRM, real samples P35S, Tnos, M. Minunni