Novel Surface Plasmon Rensonance DNA Sensing Techniques

The research of nucleic acids which includes DNA and RNA is the key to explain many life phenomena, and has become one of the most important research fields in life sciences. Generally, in DNA biosensors, the immobilized nucleic acids probes of known sequence hybridized with target DNA and such hybridization could be characterized using some physical signals which were easy to be measured. Thus, some information about target DNA, such as concentration and sequence, could be obtained by DNA biosensors. Since DNA biosensors are high-speed, sensitive, simple to operate and low pollution, they have played important roles in the field of nucleic acids research and application, such as mutation detection, gene screen, gene diagnosis, drug discovery and development, etc. At the same time, these fields have required DNA biosensors to become more sensitive, more selective and high throughout. Miniaturization and intelligentization are also challenges to DNA biosensors.In this thesis, the principle, application and the development of some common DNA biosensors were first summarized, and then investigations of novel DNA biosensors have been performed based on surface plasmon resonance (SPR) technology as follows:(1) Enhanced surface plasmon resonance for high sensitive DNA detection with the catalytic growth of Au nanoparticlesThe catalytic growth of Au nanoparticles was effective technology for improving the sensitivity. However, the deposition of metal on Au film in the process of the catalytic growth of Au nanoparticles could result in the enhancement of background. In order to solve this problem, a 25nm thick SiO₂ layer was vapor-deposited on the gold film in this work. With the low background achieved by SiO₂-coated Au films, sensitive detection of DNA hybridization using the catalytic growth of Au nanoparticles enhanced SPR was demonstrated. The detection limit of the catalytic growth of Au nanoparticles enhanced SPR was 4.8 pM complementary DNA (cDNA), which was about 250 times lower than that of direct SPR measurement (based on SiO₂ layer covered Au film). The work described here not only offered a new method to enhance the sensitivity of SPR biosensors, but also was a general method which could expand to other analytical technology which used the metal matrix.(2) Enhanced surface plasmon resonance for high sensitive DNA detection based on layer-by-layer (LBL) assembly filmsPolyelectrolyte multilayer films of (PAH/PSS)₃, which was modified on the Au surface through LBL assembly technique, could reduce significantly the metal deposition on Au surface. The preparation of such polyelectrolyte multilayer was simple, mild and cost-effective processes, without special instruments and operator. With the low background achieved by the LBL assembly film of (PAH/PSS)₃, sensitive detection of DNA hybridization using the catalytic growth of Au nanoparticles enhanced SPR was demonstrated. The detection limit of the catalytic growth of Au nanoparticles enhanced SPR was 1.8 pM cDNA, which was about 56 times lower than that of direct SPR detection (based on LBL modified Au film). The work described here provided a convenient technique to construct high sensitive biosensors.(3) Electrical switching of DNA monolayers investigated by surface plasmon resonanceIn this work, a capacitor-like cell consisted of Au film and ITO glass was designed to avoid the alternation of SPR signal induced by the electric field. The results showed that the immobilized DNA monolayers could stand straight up or lie flat down depending on the charge of Au surface. Such switching of DNA monolayers could be influenced by the strength of electric field and surface coverage of DNA probes. Meanwhile, it was found that DNA hybridization efficiency could be enhanced or decreased when DNA probes stood straight up or lay flat down on the gold surface, depending on the potential of gold substrate. Since a number of biomolecules are electrosensitive, this work may provide a unique design feature for the construction of more sensitive biosensors, biochips and nanoscale electronic devices. In addition, this work could offer a new method for the research of functional surface, such as the controllable, reversible switching surface.(4) Recognition of single-base mismatch DNA by electroelution in the presence of Au nanoparticlesSince single base mismatch DNA hybridization could generate signal, it could disturb the detection of cDNA and result in false positive signal. Here, signal caused by single base mismatch DNA could be obviously reduced by eletroelution in the presence of Au nanoparticles, while signal caused by cDNA would not be influenced. This method was simple and convenient. Probes used here was simple to preparation, low-cost and easy to popularize. This work provides a new method not only for the recognition of single-base mismatch DNA, but also for the construction of high selectivity biosensors.(5) High sensitivity and selectivity surface plasmon resonance DNA biosensors based on electroelution and the catalytic growth of Au nanoparticles.The effect of electric field on the stability of (PAH/PSS)₃ on Au films was investigated first. The results showed that (PAH/PSS)₃ modified on Au surface could not be influenced by electroelution. Then, the sensitivity of such DNA biosensors was improved by the catalytic growth of Au nanoparticles, the signal caused by single-base mismatch DNA could reduced by electroelution in the presence of Au nanoparticles. The results showed that the simple and convenient method not only could detect cDNA with high sensitivity, but also avoid the influence induced by single-base mismatch DNA. This work provided a useful technique for the analysis of complex samples.