One dimensional Polyanine (PANI) nanomaterial is widely studied because of its good conductivity, chemical stability and potential application in DNA biosening technology. However, the PANI biosensor is not good enough for practical use since the sensitivity and selectivity is only in about pM level. One way to improve the sensitivity is to amplify the signal in hybridization events. Therefore a PANI nanotube array based signal enhancing detection for DNA hybridization has been designed to amplify the signal of hybridization events. The PANI nanotube array on biosensing electrode was prepared by template method.
In this dissertation, a nanoporous alumina template with pores in diameter about 55 nm was prepared through two-step anodization with anodization voltage of 80 V. The morphology could be controlled by parameters during anodization such as voltage, kind of acid, acid concentration and temperature. The effect of dissolution time in phosphoric acid on template was examined in detail and the results showed that the ordered nanoporous structure of template was destroyed when the dissolution time in 0.5 M phosphoric acid was longer than 6 hours.
We prepared PANI nanoarray using alumina nanoporous template, and discussed the mechanism of polymerization of aniline in nanoscale pores. The effect of polymerization time on PANI nanoarray was also investigated. The results showed that the PANI synthesized using template method for 1-10 minutes polymerization time demonstrated a highly orientied array structure. Then we developed a template assisted method to integrate the PANI nanotube array on electrode, The alumina template was constructed on electrode through anodization of the aluminum thin layer deposited on electrode. The template was then employed to directly fabricate PANI nanotube array on electrode. After removing the PANI film on template surface using alumina powders, the PANI nanoarray was integrated on electrode by dissolving the alumina template using H2SO4 acid.
Through immobilization of DNA probes on PANI nanoarray electrode, the electrode was converted to a DNA biosensor. We measured the sensitivity and the selectivity of the biosensor. The results showed that the electrochemical signals of DNA hybridization indicator were highly related with the target concentration from 7.557 fM to 755.7 fM with linearity of R = 0.99. The detection limit of the PANI nanoarray DNA electrochemical biosensor was 1 fM which was three orders of magnitude higher than traditional PANI biosensor. Because the sample used in our experiments was 300 l, the corresponding absolute detection limit was 300 zmol target molecules.
The high sensitivity could be contributed to two factors. First, the PANI nanoarray had much higher effective area than biosensor based on PANI particle film and non-oriented PANI nanofiber. In detection, each PANI nanowire could immobilize more than 105 probes and worked like a collecting nanodevice for hybridization signals. Secondly, the PANI nanotube array prepared using template method had high uniformity which improved the efficiency of nanodevices.
The reproducibility of the biosensor was also investigated, and the results of three repeated tests demonstrated a good reproducibility with relative standard deviation of only 3%. At the same time we examined the ability of the biosensor to discriminate the one mismatch in oligonucleotides under ultralow 35.79 fM. The measurements proved that the PANI nanoarray based DNA biosensor could distinguish one nucleotide mismatch in 21-mer target nucleic acid. This capability indicated a potential application of PANI nanoarray based DNA biosensor in single nucleotide polymorphism analysis.
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