Studies On Biosensing Analysis Based On Enzyme Assisted Multiple Cycle Amplification And Application In Detection Of Cancer Biomarker

Early detection of cancer is vital for the successful treatment of the disease. Hence, a rapid and sensitive diagnosis is essential before the cancer is spread out to the other body organs. In this thesis, a series of novel biosensing assay systems were developed for ultrasensitive detection of cancer biomarker based on aptamer-target recognition, bio-bar-code nanoprobe enhancement and enzyme assisted multiple cycle amplification strategy. The proposed methods were also implemented in the practical determination of realistic biological samples. These results demonstrated the high potential of the developed sensor devices as platforms for clinical cancer diagnosis and prognosis. The detailed content was described as follows:1. A novel approach for thrombin detection was developed by the combination of rolling circle amplification (RCA) reaction with surface plasmon resonance (SPR) array. An aptamer-based sandwich assay was performed on gold substrate. The primary aptamer could specifically capture thrombin and the hairpin structure of aptamer capture probe could be used to effectively reduce the nonspecific adsorption. The second aptamer served as a template for the ligation of a padlock probe, and the circularized probe could in turn be used as a template for RCA to produce thousands of repeated oligonucleotide sequences. Then, the secondary aptamer domain of the products bound to the immobilized protein in a classic sandwich assay format to localize the long single-strand DNA directly to the thrombin. Thus, a large amount of Au NP labeled bio-bar-coded probes could be linearly and periodically assembled to the RCA products for amplification of recognition event. The enhancement of the SPR signals was achieved by increasing surface mass and refractive index from clustered AuNP conjugates on the SPR chip. The detection limit of0.78aM could be achieved. Based on hairpin aptamer capture probe and dual-aptamer recognition system, the excellent selectivity was also obtained. The high sensitivity and selectivity of RCA-based SPR aptasensor would make it a potential tool for proteomics research and clinical diagnostics.2. A cascade signal amplification strategy was proposed for detection of specific DNA sequences and Ramos cells by combining aptamer-based target-triggering strand displacement amplification (SDA) with rolling circle amplification (RCA) technique, magnetic nanoparticles (MNPs) bio-bar-code probe and surface plasmon resonance detection. MNPs acted as both DNA molecular carriers and the amplification intermediary for DNA amplification. Bio-bar-code MNPs were functionalized with the primer DNA of the SDA reaction and the primer DNA of the RCA reaction. The RCA product containing tandem-repeat sequences could serve as excellent template for periodic assembly of AuNPs which presented per cell recognition event to numerous AuNP tags for SPR signal response. Both the two-stage amplification reaction and the MNPs bio-bar-code technique showed remarkable amplification efficiency, very little nonspecific adsorption, and low background signal. The detection of specific DNA sequences and Ramos cells as low as1fM and76cells were achieved, respectively. The proposed cascade signal amplification strategy would become a powerful tool for early diagnosis of disease.3. A simple and sensitive method for the detection of lysozyme was developed based on aptamer-based target-triggering triple-amplification system. Via combining an aptamer with a common hairpin DNA probe, the homogeneous parallel amplifica-tion reactions were performed, target molecule displacement polymerization amplification (TDPA) and strand nicking-polymerization amplification (SNPA). On the basis of TDPA and SNPA signal amplification, the SPR signal was dramatically enhanced. This detection method exhibited excellent specificity and high sensitivity with a detection limit of76fM and a detection range of more than2orders of magnitude. Moreover, this detection method exhibited impressive simplicity, convenience, and rapid without multiple separation and washing steps. Furthermore, this method might be extended to sensitive detection of a variety of biomolecules whose aptamers undergo similar conformational changes.4. An ultrasensitive surface enhanced Raman scattering (SERS) method has been designed to selectively and sensitively detect the specific DNA sequences and lysozyme. The gold chip as detection substrate, the aptamer-based target-triggering cascade multiple cycle amplification and gold nanoparticles (AuNPs) bio-barcode Raman probe enhancement on the gold substrate were employed to enhance the SERS signals. The cascade amplification process consisted of nicking enzyme signaling amplification (NESA), strand displacement amplification (SDA), and circular hairpin assisted exponential amplification reaction (HA-EXPAR). With the involvement of an aptamer-based probe, two amplification reaction templates and a Raman probe, the whole circle amplification process was triggered by the target recognition of lysozyme. The products of upstream cycle (NESA) could acted as the "DNA trigger" of the downstream cycle (SDA and circular HA-EXPAR) to generate further signal amplification, resulting in the immobility of abundant AuNPs Raman probes on the gold substrate."Hot spots" were produced between the Raman probe and the gold film, leading to significant SERS enhance-ment. This detection method exhibited excellent specificity and sensitivity towards specific DNA sequences and lysozyme with the detection limit of72aM and1fM, respectively. Moreover, The practical determination of lysozyme in human serum demonstrated the feasibility of this SERS approach in the analysis of a variety of biological specimens.