DNA BIOSENSORS Hybridization indicator (bacteria , virus , genetic inherited diseases) Trace measurements of pollutants (intercalators,

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Presentation transcript:

DNA BIOSENSORS Hybridization indicator (bacteria , virus , genetic inherited diseases) Trace measurements of pollutants (intercalators, binders of DNA) Biosensing of drugs

Sequence-specific Hybridisation Biosensors Hybridisation Biosensors are used for identifying specific target sequences determining the order of the four bases : A, C, G, and T. DNA base pairing represents the basis for such hybridisation assays.

Applications of DNA Biosensors The detection of specific DNA sequences is of significance in many areas including clinical, environmental and food analysis. The analysis of gene sequences and the study of gene polymorphisms play a fundamental role in rapid detection of genetic mutations, offering the possibility of performing reliable diagnosis even before any symptoms of a disease appear. In the environmental and food areas the detection of specific DNA sequences can be used for the detection of Genetically Modified Organisms or Pathogenic bacteria.

Procedure for immobilization of DNA probe: Biosensor Assembly: Procedure for immobilization of DNA probe: Screen Printed Gold surface Probe (25-mer) 1 µmol/L overnight HS-(CH2)6-5’-GGC-CAT-CGT-TGA-AGA-TGC-CTC-TGC-C-3’ MCH (HS-(CH2)6-OH) 1 mM for 1 h S OH

Measurement of the Hybridization Reaction: Hybridization (20 min) with biotinilated sequence Gold electrode modified with DNA/MCH Interaction with Alkaline Phosphatase-Streptavidin (20 min) S= Non elettroactive P= Elettroactive S P S P S P S Electrochemical measurement Incubation with substrate (20 min)

-naphtol (elettroactive) Substrate: OH PO 4 3- + Prodoct: -naphtol (elettroactive) Substrate: -naphtyl phosphate (non elettroactive) ALKALINE PHOSPHATASE Voltammetry: Probe Differential Pulse Voltammetry (DPV) : Interval time: 0.05 s Modulation amplitude: 0.070 V Step potential 0.005 V Modulation time 0.15 s Scan potential: 0 V +0.60 V

Calibration Curve: Linear range up to 25 nmol/L Riproducibility of the measurements (n=4): 12% Detection Limit: 0.25 nmol/L High selectivity for non complementary sequence

Officially established method on European scale Detection of two regulatory sequences commonly found in transgenic plants Promoter region (35S) of the CAMV (cauliflower mosaic virus) ribosomal RNA NOS terminator of the nopalin synthase gene from the soil bacterium Agrobacterium Tumefasciens

Potential driven adsorption: +0.5 V vs Ag-SPE DNA immobilisation Potential driven adsorption: +0.5 V vs Ag-SPE Phosphate group Screen printed electrode

Samples pretreatment: filtering (0.45 µM) Influent after primary settlement Raw influent Effluent 1, 4: Raw influent 2, 5: Influent after primary settlement 3, 6: Treated water

Waste Water Analysis Florence Treatment Plants

Waste Water Samples : Influents

Conclusion Electrochemical DNA biosensor is a suitable tool to evidence the presence of cancerogenic compounds in real samples. It is unable to distinguish single compounds but it could be useful for screening purposes to evaluate total toxicity of water samples.