Study Of Chemiluminescence DNA Biosensor Based On Nanoparticles-Labled

The main idea of the reseach was to explore a novel and sensitive biosensor for the determination of DNA sequence based on the combination of flow injection chemiluminescence (FI-CL) technology, nanophase materials and DNA hybridization technology. 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. A novel and sensitive biosensor for the determination of short sequence of DNA based on flow injection chemiluminescence system of luminol-H₂O₂-Cu²⁺ was developed in the present work. The DNA probe labeled with copper sulfide nanoparticles (CuS NPs) could hybridize with target DNA immobilized on glass-carbon electrode (GCE). The hybridization events were monitored by the CL intensity of luminol-H₂O₂-Cu²⁺ after the cupric ions was dissolved from the hybrids. A preconcentration process of cupric ions was performed by anodic stripping voltammetry (ASV) technology to improve the sensitivity of the biosensor. Under the optimum conditions, the CL intensity was proportional to the concentration of target DNA in the range of 2.0×10^-12 to 1.0×10^-10 mol/L. A detection limit of 5.5×10^-13 mol/L of target DNA was achieved. The CL intensity of two-base mismatched sequences and noncomplementary sequences were also detected. The experiments indicated that two-base mismatched sequences showed weaker CL intensity and noncomplementary sequences gave no response at all. The results indicated that the DNA biosensor designed in this method possessed excellent selectivity. Finally, the possible reaction mechanism of CL luminol-H₂O₂-Cu²⁺ system used in the whole experiments was reserched systemically, and a proper explanation was proposed.2. A novel and sensitive flow injection chemiluminescence assay for the sequence specific DNA detection based on the signal amplification with nanoparticles was developed on the basis of our preceding work. The"sandwich-type"DNA biosensor was fabricated with the thiol-functionalized capture DNA probe firstly immobilized on Au electrode and hybridized with one end of target DNA, the other end of which was recognized with signal DNA probe labeled with CuS and Au NPs on the 3'-and 5'-terminus, respectively. The hybridization events were monitored by the CL intensity of luminol-H₂O₂-Cu²⁺ after the cupric ions were dissolved from the hybrids. We demonstrated that the incorporation of Au NPs in this sensor design significantly enhanced the sensitivity and the selectivity because a single Au NP can be loaded with hundreds of signal DNA probe strands which were modified with CuS NPs. A preconcentration process of cupric ions performed by anodic stripping voltammetry technology further increased the sensor performance. As a result of these two combined effects, this DNA sensor could detect as low as femtomolar target DNA and exhibited excellent selectivity against two-base mismatched DNA. Meanwhile, the optimum conditions were chosed, and charactor of DNA probe were also reserched systemically. Under the optimum conditions, the CL intensity was increased with the increase of the concentration of target DNA in the range of 2.0×10^-14 to 2.0×10^-12 mol/L A detection limit of 4.8×10^-15 mol/L of target DNA was achieved.3. We developed a new approach for flow injection chemiluminescence detection of single-nucleotide polymorphism using CuS NPs. In Preparetion. The main purpose of the biosesor was to detect DNA mutation and DNA damnification. The principle could be summarized as follows: The DNA biosensor was fabricated with the thiol-functionalized capture DNA probe firstly immobilized on Au electrode and hybridized with single-base mismatched target DNA, the unreactable capture DNA probe was hybridized with complementary DNA to block all active reaction sites, respectively. The recognization of target DNA was based on DNA polymerase I (Klenow fragment)-induced coupling of the CuS NPs modified guanine probe to the mutant sites of duplex DNA under the Watson-Crick base pairing rule. The hybridization events were monitored by the CL intensity of luminol-H₂O₂-Cu²⁺ after the cupric ions was dissolved from the hybrids. A preconcentration process of cupric ions was performed by anodic stripping voltammetry technology to improve the sensitivity of the biosensor. Theoratically, if there was no mismatched bases in a sample, probe could not couple with dsDNA, which would give no response at all. Under the optimum conditions, the CL intensity increased with the concentration of target DNA, which proved the feasibility of the DNA biosensor.