Immobilization, dsDNAs

Next, some typical examples will be presented of how a DNA-electrochemical biosensor is appropriate to investigate the DNA damage caused by different types of substances, such as the antioxidant agent quercetin (Scheme 20.1), an anticancer drug adriamycin (Scheme 20.2) and nitric oxide. In all cases, the dsDNA damage is detected by changes in the electrochemical behaviour of the immobilized dsDNA, specifically through modifications of the purinic base oxidation peak current [3,5,40]. 

Immobilize dsDNA in different concentrations onto the pretreated PGEs by using dip-coating procedure. For this procedure, five PGEs were dipped into the same vial containing 150 pL dsDNA prepared in ABS during 1 h. 

Toxic aromatic amines which constitute a very important class of environmental pollutants can be easily detected by electrochemical DNA biosensors. The electrochemical biosensing strategy was developed by Wang s group based on the intercalative behavior of aromatic amines onto an immobilized dsDNA layer... 

A chitosan-modified CP (ChiCP) material was prepared for the electrostatic adsorption of dsDNA, ssDNA and ODNs [92]. The immobilized ODN could selectively hybridize with the target DNA to form a hybrid on the ChiCP surface. 

Electrochemical impedance measurements of the physical adsorption of ssDNA and dsDNA yields useful information about the kinetics and mobihty of the adsorption process. Physical adsorption of DNA is a simple and inexpensive method of immobilization. The ability to detect differences between ssDNA and dsDNA by impedance could be applicable to DNA biosensor technology. EIS measurements were made of the electrical double layer of a hanging drop mercury electrode for both ssDNA and dsDNA [34]. The impedance profiles were modeled by the Debye equivalent circuit for the adsorption and desorption of both ssDNA and dsDNA. Desorption of denatured ssDNA demonstrated greater dielectric loss than desorption of dsDNA. The greater flexibility of the ssDNA compared to dsDNA was proposed to account for this difference. 

Polypyrrole has the potential to provide an effective method for reagentless transduction by immobilization of the ssDNA probe within the polymer matrix. Significant differences in the impedance profile of ssDNA and dsDNA have been demonstrated [59]. The differences in the impedance profile are purportedly based on intercalation differences of the polymer with ssDNA compared to dsDNA. The exact mechanism for impedimetric change resulting from conducting polymer films has not been identified, although it is likely linked, much like the impedimetric response of pure DNA, to the change in ion density that accompanies the double strand compared to the... 

Although several different DNA adsorption methods have been used on different types of electrodes [19,25], the immobilization of dsDNA to... 

In order to compare the reproducibility and efficiency of dip-coating procedure used for DNA immobilization separately onto five pretreated PGEs in one vial, the same experiments was repeated in different immobilization times by using each pretreated PGE as dipping separately into each vial containing dsDNA at 16 pg/mL concentration level. 

The electrochemical sensing of calf thymus dsDNA and ssDNA was performed in this study by using DPY with a disposable graphite sensor, PGE. dsDNA or ssDNA was immobilized onto pretreated PGEs by dipcoating procedure, the oxidation signal of guanine was then measured. 

After dsDNA in different concentrations as 1, 2, 4, 8, 16 and 32 pg/ mL was immobilized onto the pretreated PGEs followed by dip-coating procedure during lh, there was an increase observed at guanine oxidation signal gradually (shown in Fig. 27.1A) in parallel to increasing concentrations of dsDNA. 

Five subsequent experiments performed for electrochemical detection of DNA at concentration level of dsDNA as 16 pg/mL immobilized onto PGE during 1 h gave reproducible results with a mean guanine signal of 885 nA and an RSD value of 6.92% (n — 5). The detection limit (DL) estimated from S/N = 3 corresponds to 400 ng/mL dsDNA concentration in the 150 pL samples. 

The changes at guanine oxidation signal obtained in different immobilization time from 7.5 to 120 min and at 16 pg/mL concentration level of dsDNA are shown in histogram in Fig. 27.2. It can be concluded that 7.5 min was found as the optimum immobilization time for DNA onto PGE surface resulting in reproducible results with an RSD% (n — 5) of 2.67%. There has also not been observed any significant difference batch-to-batch performed in different vials under same conditions (not shown). 

In order to compare the reproducibility and efficiency of dip-coating procedure used for DNA immobilization separately onto five pretreated PGEs in one vial, the same experiments were repeated in different immobilization times from 7.5 to 30 min by using each pretreated PGE dipped separately into each vial containing dsDNA at 16pg/mL concentration level (shown in Fig. 27.3). In comparison to results obtained by former procedure of five PGEs in one vial, here the better reproducibility was observed with RSD% (n — 5 vials containing each of them as one PGE) of 1.51%. 

Fig. 27.2. Histogram showing the changes at guanine oxidation signal obtained in different immobilization time from 7.5 to 120 min and at 16 gg/mL concentration level of dsDNA. Other conditions are as in Fig. 27.1A.

Fig. 27.3. Histogram showing the changes at guanine oxidation signal obtained in different immobilization time from 7.5 to 30 min and at 16 gg/mL concentration level of dsDNA. Pretreatment of PGE surface by applying +1.4V for 30 s in ABS dsDNA immobilization by dip-coating procedure during 1 h for each PGE separately in one vial DPV measurement by scanning from +0.20 to +1.40 V at 50 mV pulse amplitude and 30 mV/s scan rate.

Determination of the optimal experimental conditions for the atomic force microscopy (AFM) characterization of the surface morphology of a DNA electrochemical biosensor obtained using different immobilization procedures of calf-thymus double-stranded DNA (dsDNA) on a highly oriented pyrolytic graphite (HOPG) electrode surface. 

The electrochemical study of the in situ interaction of quercetin, adriamycin, DETA/NO and their metabolites with double-stranded DNA (dsDNA) immobilized on a glassy carbon electrode (GCE) surface. 

Fig. 30.3. Evaluation of the efficiency of the dsDNA denaturing treatment. (A) Heating treatment of the dsDNA before immobilization. (B) Heating treatment of the dsDNA before immobilization plus denaturing alkaline treatment after immobilization. 153.8 ng of poly(dA)poly(dT) as DNA target 9.97 pmol dT(50)-biotin and 9.00 /ig of enzyme conjugate. The nonspecific adsorption signal is shown in black (more details in Ref. [2]).

---