Study Of DNA Biosensors Based On A Doped Screen-printed Electrode With Polyaniline Nanotubes And Room-temperature Ionic Liquid

The main idea of the reseach was to modified the commercial graphite ink (Electrodag 423SS) for manufacture of high sensitivity, high selectivity, single-used and stable screen-printed DNA sensor, which also determinated DNA sequence and tumor cells. It provided a basic theoretical reseach for clinic diagnoses of many diseases at early stage. In this paper, we prepared three types of DNA biosensors which could be summarized as follows:1. Preparation of modified screen-printed electrode (SPE). Polyaniline nanotubes (PANINTs) with appropriate shape, size and conductivity were synthesized, and were doped into the commercial graphite ink together with room-temperature ionic liquid (RTIL) or the printing of the SPE, and chitosan was covered on the electrode surface. A homogeneous, stable and highly conductive electrode surface was thus obtained. The optimal amounts of PANINT, RTIL and chitosan were investigated. 2. The DNA assay based on the SPE described above was established. The target DNA sequences were specifically recognized by the capture probes immobilized on the SPE surface and the reporter probes labeled with HRP through avidin-biotin binding. o-Phenylenediamine was catalyzed by HRP to produce 2,3-diaminophenazine, which generate electrochemical response. The target DNA could be quantitatively detected in the range from 8×10-13-2×10-14 M and a detection limit of 8.37×10-15 M was found for this sensor. The signal for the complementary DNA was much larger and apparently distinguishable from the control signal or the mismatched sequence signal. A good selectivity was thus obtained on this sensor.3. A novel and sensitive DNA sensor for the sequence specific DNA detection based on the signal amplification with gold nanoparticles and hexaamineruthenium(III) chloride (RuHex) was developed on the basis of our preceding work. A DNA detection protocol was established based on this sensor in sandwich hybridization mode. Au nanoparticles were bound to the hybridization complexes through reporter probes, and RuHex was attached to the bar-code DNA on nanoparticles. This sensor was found to be able to distinguish the complementary target DNA at low concentration and the three-base mismatched DNA at higher concentration. The target DNA could be quantitatively detected in the range from 1.0×10-16 M to 1.0×10-14 M, and a detection limit of 8.0×10-17 M was found.4. A new approach for the detecting of tumor cells, Ramos was established based on aptamer-based cell recognition and the isothermal circular DNA strand-displacement polymerization. This assay was carried out in two steps. A hybridization complex of the aptamer and its partially comlementary sequence ("messenger sequence") were immobilized on the magnetic beads. The recognition of Ramos cells on this complex resulted in the release of the messenger, which triggered a strand-replacement DNA replication for itself, resulting in the multiplication of its concentration. The messenger hybridized with hairpin probes on the electrodes to open their stem parts, to which a gold nanoparticle with primer sequences and bio-bar-code sequences was hybridized, and an isothermal DNA polymerization was initiated, which replaced the intermediary target sequence and released it into the solution. The released messenger sequence found another hairpin probe to begin another strand-replacement polymerization. The signal was generated through the chronocoulometric interrogation of RuHex that was attached on the bio-bar-code sequences. A detection limit of 1.2×10-17 M was found for the DNA sequence detection, and 100 cells for cell detection. This sensor was found to be able to distinguish the complementary target DNA at low concentration and the three-base mismatched DNA at higher concentration. Selectivity in the cell detection was thus obtained on this sensor. The optimal experimental conditions were also explored.