DNA BIOSENSORS FOR FOOD SAFETY and ENVIRONMENTAL MONITORING.pdf

DNA BIOSENSORS FORFOOD SAFETY andENVIRONMENTALMONITORINGUniversit degli Studi di Firenze(ITALY)Dipartimento di ChimicaLaboratorio BiosensoriMarco MasciniMasciniunifi.itwww.chim.unifi.it/ana/“Biosensors are analytical devices incorporating abiological material(e.g.tissue,microorganisms,organelles,cell receptors,enzymes,antibodies,nucleic acids etc.),abiologically derived material or biomimic intimatelyassociated with or integrated within a physicochemicaltransducer or transducing microsystem,which may beoptical,electrochemical,thermometric,piezoelectric,ormagnetic.”Biosensors usually yield a digital electronic signal which isproportional to the concentration of a specific analyte orgroup of analytes.While the signal may in principle becontinuous,devices can be configured to yield singlemeasurements to meet specific market requirements.”(One-shot biosensors)Biosensors&Bioelectronics(2000)?Trace measurements of pollutans (intercalators,binders of DNA)?Hybridization indicator(bacteria,virus,genetic inherited diseases)?Biosensing of drugs?Disposable electrochemical biosensors forthe detection of low molecular weightsubstances which show affinity with DNA?Electrochemical DNA biosensors for thedetection of specific DNA sequencesElectrochemical DNA biosensorScreen printed electrodesDNA immobilizationElectrochemical interrogation of the surfaceAnalysis of standard solutionsReal samples analysis(in situ)Comparison with reference methodScreening analysis of toxic compounds in environmental samplesScreen printed electrodes(SPE)1 cmInsulating inkGraphite counter electrodeSilver pseudo-reference electrodeGraphite working electrodeElectric connectionsAdvantages:each electrode is disposable:no contamination easy to produce and to use low costPortable apparatus(PalmSens)Palm Sens?&pocket PCScreen printed electrodesStirrer plateDNA biosensorThe system is based on a screen printed electrochemical cell,and a PalmSens instrument for electrochemical analysis connected with a pocket PC.Potential driven adsorption:+0.5 V vs Ag-SPEScreen printed electrodePhosphate groupElectrochemical procedure Pretreatment:+1.6V for 120s and+1.8V for 60s,in 0.25M aceticbuffer(pH=4.75)with 10mM KCl,under stirred conditions.DNA immobilisation:50ppm of double stranded calf thymus DNA,+0.5 V for 120s,under stirred conditions.Blank or sample interaction:10l of the sample solutions onto theworking electrode surface for 2 min.Measurements:Square wave voltammetry in 0.25M acetic buffer(pH=4.75)with 10mM KClParameters:potential range from+0.2V to+1.45V Frequency=200Hz,Estep=15 mV,Eamplitude=40 mVAnalytical signalSb:guanine peak after the interaction with the blank solutionSs:guanine peak after the interaction with the sampleGenoxicity index:S guanine%=(Ss/Sb)*100Guanine OxidationEp=+1.0 V vs Ag-SPEThe interaction of the DNA with pollutant moleculesmodifies the oxidationof the DNA guanine base0.20.40.60.81.01.21.40.000.751.502.25 Blank Analytei(A)E(V)SbSsNH2NH22-Naphthylamine and 2-Anthramineon dsDNA Calf Thymus biosensor0510152025020406080100120R%2-Naphthylamine(M)0.00.40.81.21.6020406080100R%2-Anthramine(M)NNNAcridine orange on dsDNACalf Thymus biosensor0.00.10.20.30.420406080100R%Analyte(M)0.60.81.01.20.00.20.4Guanine peakAnalyte peak Phosphate buffer Acridine Orange 0.05 M Acridine Orange 0.10 M Acridine Orange 0.20 Mdt/dE(s/V)E(V)Standard compounds*Solution containing 1%of DMSODNA biosensorR%DNA biosensorR%2-Anthramine(0.077?g/ml)-39 10%Stearylamine(13 g/ml)-14 3%2-Anthramine(0.116?g/ml)-56 13%Stearylamine(46 g/ml)-42 6%2-Anthramine(0.154?g/ml)-67 11%Mix of polyethoxylates(0.1 g/ml)-9 9%1,2-Diaminoanthraquinone(0.019 g/ml)-49 6%Mix of sulfonates(1 g/ml)-23 11%1,2-Diaminoanthraquinone(0.039 g/ml)-86 9%Estradiol+Ethynylestradiol(0.0005?g/ml)-37 2%2-Aminonaphtalene(0.82 g/ml)-39 5%2-Chlorophenol(10?g/ml)-39 11%2-Aminonaphtalene(3.26 g/ml)-68 9%4-nitroquinoline-N-oxide*(0.5 g/ml)-70?3%Acridine orange(0.015 g/ml)-20 6%4-nitroquinoline-N-oxide*(0.01 g/ml)-50?8%Acridine orange(0.058 g/ml)-62 8%N-methyl-N-nitro-nitrosoguanidine(40 g/ml)*-20?10%Mix of LAS(1 g/ml)-26 5%N-methyl-N-nitro-nitrosoguanidine(4 g/ml)*-5 3%Mix of LAS(5 g/ml)-64 7%Carbaryl(0.002?g/ml)-48 7%Bioluminescence as an indicator of toxicity:Toxalert SystemLuminescent bacteria,e.g.Vibrio Fischeri NRRLB-11177,naturally emit light,calledbioluminescence.The enzyme involved is bacterial luciferase which catalyses the followingreaction:FMNH2+O2+R-CO-H?FMN+R-COOH+H2O+LightBioluminescence is directly linked to the vitality,themetabolic status,of the cell.A toxic substance will causechanges to the cellular state which are rapidly reflected in adecrease in bioluminescence.The reduction of bioluminescence can be measured witha photomultiplier in a luminometer.Reference method(ToxAlert 100,MERCK)Cytotoxicity testBioluminescent bacteria (Vibrio Fischeri):FMNH2+O2+RCHO FMN+RCOOH+H2O+light(?=489 nm)luciferaseReconstitution of Liquid Dried Bacteria(15)Preincubation of bacteria suspension(500?l;15)Measurement of luminescence;addiction of 500?l of sample solutionIncubation(30)Measurement of the remaining luminescenceToxicity index:inhibitory effect(I%):I%=Ib-IsIb100I%?20Toxic sampleProcedure:Ib=Luminescence intensity in the blank solutionIs=Luminescence intensity in the sample solutionSample(1 liter)Filtering(0.45?m)SPE(C18)Eluition(AcOEt;NaCl 2%)Preconcentration:160 timesRiver water samples(Arno)123456789020406080100020406080100-R%(DNA biosensor)ToxAlert 100 DNA biosensorI%(ToxAlert 100)SamplesWaste Water AnalysisFlorence Treatment Plants012345678910 11 12 13 14 15020406080100020406080100I%(ToxAlert 100)-R%(DNA biosensor)ToxAlert 100 DNA biosensorRaw influentsWaste Water Samples:InfluentsRole of DNA biosensors for RiskAssessment in marineenvironment?Monitoring of DNA binding substances in tissues or biological fluids of organisms not exposed to pollutants tissues or biological fluids of organisms exposed to pollutants organisms tissues or biological fluids spiked with pollutants?Comparison with bio-markers(i.e.weight,growth of theorganisms)?Comparison with other molecular bio-assays(i.e.Cometassay)What are PAHs?Sources:incomplete combustion of organic material likecoal and oil;forest fires and volcanic eruptions.PAHs and their metabolites,may intercalate betweenadjacent base pairs or may form DNA-adducts that cantrigger carcinogenic processes.Therefore,all PAHs aremutagenic and harmful to reproduction.The heavyPAHs(five to seven rings)in particular,are a majorcause of these chronic damages.Due to their liposolubility they are absorbed fairly easilyby living organisms,and can be accumulated ortransformed into more toxic substances.The detection of PAH metabolites in fish bile(organisms)can be used as a marker of recent exposure atcontaminated sites.Analysis of PAHs metabolitesstandard solutions02468105060708090100S%1-OH-BenzoaPyrene concentration(g/m L)02468105060708090100S%3-OH-BenzoaPyrene concentration(g/mL)OHOH1-OH-Benzo a Pyrene3-OH-Benzo a PyreneFish species Atlantic cod Live chiefly in cold or temperate northern seas,atdephts of 180-360 m Max lenght 1.5 m Commercially important as food as the source ofcod-liver oil Haddock Same family of cod Shorter Very abundant in the North Atlantic Ocean,ittl i lhl1)Karmy island area,species:Atlantic cod005060708090100AreasS%Blank 1 2 3 4 5 6 Blank&reference Polluted samples Very polluted samplesSamples diluted 1:5000 with acetic buffer 0.25M pH 4.75+1%EtOHFish from six sea areas at different pollution levelsN5243N5Km61PollutionsourceComparison with different testsFIELDSITETYPEOF SITEDNAbiosensorS%F-int 341/383Pyrene-like metab.(g/mL bile)Condition index(weight(g)xlenght(cm)-3)x100 Liversomatiticindex 1Reference100?699?53.73.50.93?0.062.0?0.82Lowcontamin.86?8*83?7*4.86.00.84?0.08*1.9?0.83Near aharbour82?7*75?1*15.0*17.7*0.84?0.08*1.4?0.5*4Moderatecontamin.80?5*86?1*56.0*61.0*0.89?0.071.7?0.85Highcontamin.75?8*72?8*158.0*154.0*0.85?0.08*1.8?1.46Highcontamin.54?2*55?6*1175.0*1049.0*0.76?0.12*1.2?0.3*Measured performed by Akvamilj Centre(Randaberg,Norway)*Significantly different from reference site(1),P0.05.2)Tampen area,species Haddock-405060708090100 exposedreferenceS%fish bile samplesFish from two sea areas at different pollution levels,sampling in 2002Samples diluted 1:5000 with aceticbuffer 0.25M pH 4.75+1%EtOH00405060708090100110 S%blank reference exposed10 Km awayfrom TampenStatfjordoil fieldAnalysis of real soil samples ACNA Cengio(SV)soil,March 2003,different depths Soil pretreatment The same as described before Extraction 0.5g soil with 20mL solvent acetic buffer 0.25M pH 4.7 phosphate buffer 25mM pH 7.4 ethanol 96%Mix 2min,then ultrasonic bath 30 min X 2(T 25C)Centrifuge 5000 r.p.m.10 min(T 25C)Filtration with filters 0.45mAliith DNA biResultsBlank1234020406080100S%Samples/areas ACNA Extract with acetic buffer 0.25M pH 4.75 Extract with phosphate buffer 25mM pH 7.4 Extract with ethanol 96%134BORMIDA RIVER2AreaDepth(cm)1202Superficial320440 Electrochemical DNA biosensor is a suitabletool to evidence the presence of compoundsof environmental interest in real samples.It is unable to distinguish single compoundsbut it could be useful for screening purposesto evaluate total toxicity of water samples.ConclusionDNA structureHybridisation Biosensorsare used for identifying specific target sequencesdetermining the order of the four bases:A,C,G,and T.DNA base pairing represents the basis for suchhybridisation assays.Sequence-specific Hybridisation BiosensorsApplications of DNA BiosensorsThe detection of specific DNA sequences is of significance inmany areas including clinical,environmental and food analysis.The analysis of gene sequences and the study of genepolymorphisms play a fundamental role in rapid detection ofgenetic mutations,offering the possibility of performingreliable diagnosis even before any symptoms of a diseaseappear.In the environmental and food areas the detection of specificDNA sequences can be used for the detection of GeneticallyModified Organisms or Pathogenic bacteria.Screen-printedScreen-printed electrochemical electrochemical sensorssensors1 cmSilver pseudo-reference electrodeGold working electrodeGraphite auxiliaryelectrodeScreen-printed electrodes sheet 2 cm3 cmStep-by-step screen-printing processFeatures of screen-printed gold electrodes(SPEs):Low cost,Reproducibility,Mass productionSingle-use sensors avoid samplecontamination and need for regenerationof the electrodic surfaceElectrochemical GenosensorsDetermination of specific sequence od DNADetermination of the Promoter CaMV 35SHydentification of geneticallymodified organismsProcedure for immobilization of DNA probe:SSSSSSProbe(25-mer)1 mol/L overnightScreen Printed Gold surfaceMCH(HS-(CH2)6-OH)1 mM for 1 hSSSSSSSOHSOHSOHSOHSOHSOHSOHHS-(CH2)6-5-GGC-CAT-CGT-TGA-AGA-TGC-CTC-TGC-C-3Biosensor Assembly:SSSSOHSOHSOHSOHSOHSOHSOHSOHSSSSOHSOHSOHSOHSOHSOHSOHSOHSSSSOHSOHSOHSOHSOHSOHSOHSOHSP SPSPSSSSOHSOHSOHSOHSOHSOHSOHSOHSHybridization (20 min)with biotinilated sequenceGold electrode modifiedwith DNA/MCHInteraction withAlkaline Phosphatase-Streptavidin(20 min)Incubation withsubstrate (20 min)Measurement of the HybridizationReaction:ElectrochemicalmeasurementS=Non elettroactiveP=ElettroactiveDifferential Pulse Voltammetry(DPV):Interval time:0.05 sModulation amplitude:0.070 VStep potential 0.005 VModulation time 0.15 sScan potential:0 V?+0.60 VPOOHOHOPO43-+OHProdoct:?-naphtol(elettroactive)Substrate:?-naphtyl phosphate(non elettroactive)ALKALINE PHOSPHATASEVoltammetry:0.00.10.20.30.40.50.60.02.55.07.510.012.515.0Ep=+0.2 V vs.Ag-SPE Sonda Com pl.200 nmol/Li(A)E(V)Probe02004006008001000120002468101214010203002468 Experimental data Linear fit i(A)nmol/L i(A)Complementare/(nmol/L)Complementare Non-complementareCalibration Curve:Linear range up to 25 nmol/LRiproducibility of the measurements(n=4):12%Detection Limit:0.25 nmol/LHigh selectivity for non complementary sequenceGMOs can be defined as organisms whose genome has been alteredin a way that does not occur naturally by mating or naturalrecombination.Introduction of foreign genes to produce novel proteins Pest&disease toleranceImprovement of the chemical profileA gene cassette consists of a promoter (P),a codingregion and a terminator(T)Gene cassettePromoter Terminator Coding regionOfficially established method on European scaleDetection of two regulatory sequences commonly found in transgenic plantsPromoter region(35S)of the CAMV(cauliflower mosaic virus)ribosomal RNANOS terminator of the nopalin synthase gene from the soil bacterium Agrobacterium TumefasciensSondaBase sequence of the PCR amplified productsPrimerPrimergctcctacaaatgccatcatTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATgacgcacaatcccactatccgaatcctgttgccggtcttgCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATAtagcgcgcaaactaggataaSonda35STNOSPrimerPrimerSequence 35S amplified with the PolimeraseChain reaction(PCR)Negative Control:sequence biotinilated with the gene of lectine,217bpTARGET:sequence of promoter CaMV 35S(195 bp)amplified from the plasmidepBI 121 extracted from E.Coli.5-GCT CCT ACA AAT GCC ATC ATT GCG ATA AAT GAA A GGC CAT CGT TGA AGA TGC CTCTGC C GAC AGT GGT CCC AAA GAT GGA CCC CAC CCA CGA GGA GCA TCG TGG AAA AAG AAGACG TTT CAA CCA CGT CTT CAA AGC AAG TGGATT GAT GTG ATA TCT CCA CTG ACG TAAGGG ATG ACG CAC AAT CCC ACT ATC-3ProbeThe amplified probes are biotinilated in 5(obtained by biotinilated primers)SSSCleaning of the amplified(with columns of acetale membranes)PROCEDURE for the ANALYSYS FOR AMPLIFIED Thermic denaturation:5 min.a+100C;5 min a 0C0204060801001200255075100125150175 i(nA)P35S/(nmol/L)Sample Conc.(nmol/L)Signal (nA)1 100 161?15 2 100 149?25 3 100 151?15 4 100 150?3 5 100 151?11 mix PCR -16?3 mix PCR -13?1 mix PCR -11?2 Neg.Control.100 6?1 Neg.Control 100 18?4 Neg.Control.100 10?5 2xSSC-7?2 Results obtained with amplified samples:Linearity in a wide range of concentrations(0-120 nM)Detection Limit(S/N=3)=15 nMHigh selectivityMOLECULAR RECOGNITION ELEMENTS?Antibodies?Receptors?Nucleic AcidsBIOLOGICAL?Molecular ImprintedPolymers(MIP)?Peptide Nucleic Acid(PNA)?Oligopeptides?AptamersBIOMIMETICAnalyticalSignalMolecularRecognitionTransductionAFFINITY BIOSENSORanalyteAPTAMERSAptamers are oligonucleotides that are identified through a combinatorial selection process for high affinity binding to target molecules.In the selection process,a combinatorial library of oligonucleotides is passed through a column containing the immobilized target.Those oligonucleotides that do not bind are discarded,while those that bind are collected and amplified.This cycle is repeated several times until a small number of affinity binders,or aptamers,have been isolated from the combinatorial pool.APTAMERs?Synthetic sequence(30-40 mer)of nucleic acids,single strand DNA or RNA,obtained by an in vitroselection