Amperometric DNA sensor using the pyrroquinoline quinoneglucose dehydrogenase /avidin conjugatea.
ChemicalsStreptavidin, biotin beads and mineral oil and N-succinimidyl-3- (2-pyridylditio) propionate(SPDP), sulfosuccinimidyl 4N -maleimidomethyl-cyclohexane-1-carboxylate (Sulfo-SMCC)and avidin gel beads, Glucose and dimethyl sulfoxide (DMSO, 1-methoxyphenazinemethosulphate (m- PMS) and carbon paste.b. Preparation of biotin-immobilized electrodes:Two milligrams of biotin beads were mixed with 20 mg of carbon paste and 40 ml of mineral oil.The resulting mixture was introduced into the hole of the carbon paste electrode (inner diameter3 mm, geometric surface area of 0.28 cm2, BAS Inc.), which was then gently rubbed with a filterpaper to produce a flat smooth electrode surface. This electrode was used to investigate thereaction of the (PQQ)GDH /avidin conjugate on the carbon paste electrode surface.
c. Target DNA and probe DNA sequencesPart of the sequence of the Salmonella virulence invA gene 4. was chosen as the target DNAsequence and it was determined by BLAST 5. to be specific for the invA gene.
The probe DNAsequence is complementary to that of the targetDNA and was immobilized onto the carbon paste electrode. The control DNA has a random DNAsequence having no relation to the target DNA and was synthesized to investigate the selectivityof the DNA electrochemical sensor. All the oligonucleotides were labeled with biotin at the 5 end,indicated as ‘bio-‘ in the sequences of the synthesized oligonucleotides shown below.Target DNA: 5′ -bio-TCGGCATCAATACTCATC- 3’Probe DNA: 5′ -bio-GATGAGTATTGATGCCGA- 3’Control DNA: 5’-bio-CTGATGAACATAC- TATCT-3’d.
Preparation of DNA-immobilized electrodeAvidin gel beads containing approximately 6.0 nmol avidin were washed with 2 /SSC buffer (30mM citric acid, 300 mM NaCl, pH 7.0), lyophilized, and then mixed with 20 mg carbon paste and40 ml mineral oil.e. Hybridization and its detection using the (PQQ)GDH /avidinconjugate.The entire detection scheme is illustrated in Fig. 1.
The DNA-immobilized electrode wasimmersed into 1 ml of reaction buffer containing target DNA or control DNA and incubated for1 h at 25 8C to allow hybridizationf. Electrochemical measurementThe working electrode (3 mm diameter, BAS Inc.), a Pt wire as counter electrode, and an Ag/AgClelectrode (Model RE-1, BAS Inc.) as reference electrode were introduced into a 10 ml waterjacketcell (BAS Inc.
Model VC-2) through holes in its Teflon cover.