Study Of Several DNA Sensors Based On Gold Nanoparticle Label

Human life is fundamentally controlled by gene. With understanding the relationship between gene and corresponding disease, it is very important to disease therapy and prognosis that the gene mutation and impressionable species are detected at molecular level. The DNA recognition technology is improved continuously with the development of molecular biology and biotechnology. In recent years DNA sensor technology is paid close attention because it has the advantages of saving detection time, analyzing the quality and quantity of samples simultaneously, as well as high sensitivity and excellent selectivity. DNA sensor technology integrates molecular biology, physics, chemistry and micro-electronic technology. It can be used in the area of disease diagnosis, contagion detection, environment monitoring, medicine research, medicolegal indentification and foodstuff analysis. According to detection methods, DNA sensors involve electrochemical sensor, optical sensor, mass sensor (for example, quartz crystal microbalance) and so on.Nowadays, it's a developing direction that nanotechnology combines with biosensor technology. Nanometerials possess unique physical and chemical properties, such as quantum size effect,small size effect,surface effect and quantum tunneling effect. Functional nanoparticles (electronic, optical and magnetic) can connect with DNA molecules to enlarge the signals of DNA detection. Specially, nanogold becomes one of the most suitable nanoparticles because of its simple preparation procedure, controlled diameter, biology compatible and easy to label with DNA.Sensitivity is one of the most important properties of DNA sensors. In this study, we attempted to improve the sensitivities of several DNA sensors via the labeling of gold nanoparticles. The ma in contents are as follows:1.Gold label silver stain method of DNA detection on polypropylene supportCompact polypropylene slices and porous polypropylene membranes were modified by the hydrogen/nitrogen plasma treatment in order to graft amino functional group (-NH2) onto the surfaces. Oligonucleotides were in situ synthesized on the aminated polypropylene support. At the same time, the Oxidizing Agent of in situ Synthesis was improved in order to develop biochip fabrication methods which were suitable for printing chemicals of DNA microarray synthesis quickly and in large scale. Gold nanoparticle labeled target DNAs were hybridized to the synthesized oligonucleotide array, and then the slices and membranes were exposed to the Silver Enhance Solution for signal amplification. It showed that the improved I2/Ac2O/AcOH/THF oxidizing agent made the in situ Synthesis more feasible by eliminating the decrease of coupling efficiency which owing to the cross reagent interfering arises from pyridine and water in the traditional I2/H2O/Pyridine/THF oxidizing agent. Using this oxidizing agent, a single step coupling efficiency of 98.2% was achieved,and the complementary and mismatched DNA could be distinguished clearly. The N2/H2 plasma treated polypropylene slices and membranes can be used as the substrate of in situ synthesis of DNA directly. The new gene-detection system, which employs a plasma treatment on polypropylene slices and a DNA in situ synthesis technique based on colorimetric detection, has a sensitivity as low as 100 fM, and can distinguish mismatch sequences clearly at this target concentration. The ratio of the background-subtracted gray-scale values for a perfect match, single-base mismatch, 2-base mismatch and 3-base mismatch is 22:16:9:4. The sensitivity of DNA spotting system was 100 pM, which was 3 orders of magnitude lower than that of DNA in situ synthesis system, and the signals of the former were about half of that of the latter under the same target DNA concentration. On the surface of amino plasma-grafted polypropylene membranes, the hybridization signals were stronger than that of the commercial polyacrylamide modified polypropylene membranes that load 0.07μmol/cm2 free primary amino functions. Complementary and mismatched sequences were clearly distinguished. Bigger nanoparticle diameter, as well as higher concentration of thiol DNA modified gold nanoparticles lead to stronger hybridizationsignals.2. Gold nanoparticle enhanced electrochemical DNA detectionCarbon nanotubes and meso-porous molecular sieve (SBA-15) were mixed with graphite powder and olefin oil respectively to fabricate carbon nanotube paste electrode (CNTPE) and molecular sieve carbon paste electrode (MSCPE). DNA probes were immobilized on the prepared electrodes by streptavidin-biotin conjugation, then hybridized with Au nanoparticle labelled target DNA to connect the first layer of colloid gold. Furthermore, the second layer of colloid gold was connected via layer-by-layer hybridization. The electrochemical transduction was employed via monitoring the DPV response of gold nanoparticles. The CV response of immobilized DNA indicated that CNTPEs were more propitious to DNA hybridization than pure carbon paste electrode (CPE) because of the larger surface area of the former. The amount of the immobilized DNA was increased with the increase of the diameters of nanotubes. The hybridization signals were enhanced with the increase of the content of nanotube in the CNTPE. However, the electrochemical signals of DNA adsorption and hybridization of MSCPE were both weaker than that of CPE. After layer-by-layer hybridization, the DPV response was more significant than that of one-layer hybridization. Complementary and mismatched DNA can be distinguished clearly in layer-by-layer method. The detection limit of one-layer hybridization was 1 nM. The detection limit of layer-by-layer hybridization was 100 pM, which was one order of magnitude higher than that of one-layer hybridization.3. Gold nanoparticle mass-enhanced piezoelectric DNA detectionPiezoelectric DNA sensor is mass-sensitive. Gold nanoparticles were labeled with hybridized DNA to increase the mass change on the surface of gold coated quartz crystal because the mass of Au nanoparticle is much greater than that of DNA molecule. Layer-by-layer connection of gold nanoparticles was performed in order to enhance the mass change more. It showed that the labeled Au nanoparicles actually increased the frequency change of quartz crystal. Two labeling methods were carried out: one is indirect labeling method in which nanoparticles are labeled with DNA after hybridization, the other is direct labeling method in which nanoparticles are labeledwith DNA before hybridization. The frequency change of the former was much greater than that of the latter. In direct labeling method, the frequency change increased with the increase of the diameter of Au nanoparticle. But the situation was opposite in indirect labeling method. When the concentration of DNA probe was 1μM and the diameter of Au nanoparticle was 5 nm, the sensitivity of one-layer hybridization was 100 fM, and the frequency change of complementary DNA: one-base mismatched DNA was 2.27:1. The sensitivity of layer-by-layer hybridization was 10 fM, and the frequency change of complementary DNA: one-base mismatched DNA was 2.13:1. It indicated the detection limit of layer-by-layer hybridization was one order of magnitude higher than that of one-layer hybridization. This layer-by-layer mass enhanced system was applied to detect the 677TT genotype of MTHFR gene. The sensitivity achieved 10 fM and the complementary and one-base mismatched DNA could be distinguished clearly. The frequency change of complementary DNA: one-base mismatched DNA was 2.1:1.