Surface Plasmon Resonance-based Nucleic Acids Detection Technology

Nucleic acids are biomolecules that play vital role in numerousprocesses in cells from the storage, transmission and expression of geneticinformation to regulatory roles, and have become one of the most hotresearch field. It is important in the field of gene diagnosis, gene screen,mutation detection and drug development by obtaining some informationabout target, such as sequence and concentration. Therefore, to developrapid, simple, sensitivity detection method is of out-most important.Generally, in nucleic acid biosensors, the immobilized probes of knownsequence on the sensor surface, and the hybridization information could becharacterized and detected using some physical signals, and nucleic acidbiosensor has become an important technology. Surface plasmon resonancebiosensor has been widely used in biomolecular interaction analysis andquantitative detection due to its label-free, simple, sensitive, goodselectivity, little sample needed, low cost and real-time measurement. Thisresearch has been performed based on surface Plasmon resonancebiosensor by integrating molecular biotechnology for nucleic acidsdetection. This dissertation includes the following two parts:1. Surface plasmon resonance DNA biosensor has been developedfor high-sensitive detection of SalmonellaA method based on surface plasmon resonance (SPR) DNA biosensorhas been developed for label-free and high-sensitive detection ofSalmonella. A biotinylated single-stranded oligonucleotide probe wasdesigned to target a specific sequence in the invA gene of Salmonella andthen immobilized onto a streptavidin coated dextran sensor surface. The invA gene was isolated from bacterial cultures and amplified using amodified semi-nested asymmetric polymerase chain reaction (PCR)technique. The calibration curve of synthetic target DNA had good linearityfrom5nM to1000nM with a detection limit of0.5nM. While with thissystem to detect E. coli and S. aureus, no significant signal was observed,demonstrating good selectivity of the method. In addition, the hybridizationcan be completed within15min, and the excellent sensor surfaceregeneration allows at least300assay cycles. The strategy proposedperforms the advantages of free label snd sensitivity general applicability toalmost all Salmonella serotypes, which has potential application inSalmonella detection.2. Surface plasmon resonance biosensor has been developed forhigh-sensitive detection of miRNAAn approach was developed for highly sensitive and specific detectionof miRNA using a surface plasmon resonance biosensor. A thiolatedcapture DNA probe was immobilized on the sensor surface to recognize thetarget miRNA, and then an oligonucleotide probe linked to streptavidin wasemployed. The use of the streptavidin caused increase in signal, improvedthe detection sensitivity by a factor of~24, and lowered the detection limitto1.7fmol. This specificity allowed to discriminat a single mismatch. Thewhole assay takes30min, and the surface of the sensor can be regeneratedat least30times. The method was successfully applied to the determinationof miRNA human total RNA samples.