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

2. 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.

3. 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.

4. 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

5. 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)

6. -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: V
Step potential V
Modulation time 0.15 s
Scan potential: 0 V V

7. 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

8. 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

9. 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

11. 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

12. Waste Water Analysis
Florence Treatment Plants

13. Waste Water Samples : Influents

16. 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.