CHAPTER 18 • Affinity Electrochemical Biosensors for Pollution Control
417
This area was compared with the area obtained when the analyte concentration in
the sample solution was zero.
In some cases, the sample solvent was up to 10% methanol to allow analyte dissolution.
18.2.3
Analysis of River Water Sample
A preconcentration step of river water was found necessary to obtain some signals
through this technique. The water samples were prefiltered through a 0.45 Ilm filter
to clarify the water. Then 11 of sample water was passed through an Isolute column
SPE. The organic compounds extracted were eluted using 500 III ethyl acetate. The
obtained samples were dried, then 10 ml of phosphate buffer containing 3% v/v methanol were added. The analysis was carried out as described before. The extracts of the
water samples were also analyzed using a standard method with a Varian 3400 gas
chromatograph coupled to a Finnigan Mat 800 ion trap detector mass spectrometer
(GC-ITDMS).
18.3
Results
18.3.1
DNA Sensor for Binding Compounds with an Affinity for DNA
Preliminary studies were performed to identify general assay conditions that affected
the electrochemical signal of the guanine oxidation peak, like ionic strength, pH, buffer
composition, DNA concentration and form (single-stranded and double-stranded).
Figure 18.2 reports the guanine peak area obtained as a function of single-stranded
calf thymus concentration. The area increases linearly with concentrations up to
20 mg rl, then levels off. This value was generally used.
Table 18.1 reports the values of the guanine peak area as a function of the stripping
oxidation current value. The area is smaller with larger currents, but the peak appeared
very broad and not reproducible (a large RSD). 6 IlA was then generally used.
The guanine peak appears very sharp in acetate buffer; the baseline at this high
potential value was lower than in other buffer. The modified electrode performance
was tested in buffer solution containing methanol up to 10% v/v, and we observed any
variation of the electrochemical signal of guanine peak. Methanol is useful for dissolving some organic compounds.
We performed several preliminary experiments for evaluating the variation of the
area of the guanine peak obtained using single-stranded or double-stranded DNA
immobilized when the sample contained different compounds of environmental interest.
Table 18.2 summarizes these experiments, showing that the guanine peak is higher
for single-stranded DNA (ssDNA); the guanine base in single-stranded DNA is obviously more readily available for oxidation than in double-stranded DNA (dsDNA).
Buffer solution: 0.2 M acetate buffer solution pH 5.0 with 10% v/v of methanol.
The area is reported with each measurement repeated four times.
417
This area was compared with the area obtained when the analyte concentration in
the sample solution was zero.
In some cases, the sample solvent was up to 10% methanol to allow analyte dissolution.
18.2.3
Analysis of River Water Sample
A preconcentration step of river water was found necessary to obtain some signals
through this technique. The water samples were prefiltered through a 0.45 Ilm filter
to clarify the water. Then 11 of sample water was passed through an Isolute column
SPE. The organic compounds extracted were eluted using 500 III ethyl acetate. The
obtained samples were dried, then 10 ml of phosphate buffer containing 3% v/v methanol were added. The analysis was carried out as described before. The extracts of the
water samples were also analyzed using a standard method with a Varian 3400 gas
chromatograph coupled to a Finnigan Mat 800 ion trap detector mass spectrometer
(GC-ITDMS).
18.3
Results
18.3.1
DNA Sensor for Binding Compounds with an Affinity for DNA
Preliminary studies were performed to identify general assay conditions that affected
the electrochemical signal of the guanine oxidation peak, like ionic strength, pH, buffer
composition, DNA concentration and form (single-stranded and double-stranded).
Figure 18.2 reports the guanine peak area obtained as a function of single-stranded
calf thymus concentration. The area increases linearly with concentrations up to
20 mg rl, then levels off. This value was generally used.
Table 18.1 reports the values of the guanine peak area as a function of the stripping
oxidation current value. The area is smaller with larger currents, but the peak appeared
very broad and not reproducible (a large RSD). 6 IlA was then generally used.
The guanine peak appears very sharp in acetate buffer; the baseline at this high
potential value was lower than in other buffer. The modified electrode performance
was tested in buffer solution containing methanol up to 10% v/v, and we observed any
variation of the electrochemical signal of guanine peak. Methanol is useful for dissolving some organic compounds.
We performed several preliminary experiments for evaluating the variation of the
area of the guanine peak obtained using single-stranded or double-stranded DNA
immobilized when the sample contained different compounds of environmental interest.
Table 18.2 summarizes these experiments, showing that the guanine peak is higher
for single-stranded DNA (ssDNA); the guanine base in single-stranded DNA is obviously more readily available for oxidation than in double-stranded DNA (dsDNA).
Buffer solution: 0.2 M acetate buffer solution pH 5.0 with 10% v/v of methanol.
The area is reported with each measurement repeated four times.