Novel Methods Of Optical Biosensors For The Detection Of Biotin And Micrococcus Enzyme Based On Nanomaterials

Along with the development and deep research of life science, measurement andanalysis of the biological information for a various kinds of biological small moleculesand enzymes which plays an important role in many biological processes have a greatsignificance of scientific research in biological medicine, clinical treatment anddiagnosis, environmental monitoring, material science, etc. Because of some uniqueperformance of nanomaterials such as surface effect, small size effect, quantum sizeeffect and macroscopic quantum tunnel effect, etc, it has been widely used in theanalysis of biological sensing technology and promoted the development of thebiological sensing technology greatly. Therefore, the work of this thesis is concernedof the main problems for the detection of biological small molecules and nucleic acidenzyme. Based on the special properties of nanomaterials which can be used as signalindicator and the platform for the analysis of the sensing technology, we have beenproposed several kinds of label-free optical biosensors which with nice sensitivity andspecificity and simplicity of operation for biotin and micrococcus enzyme detection.The main research contents of this thesis were describes detailly as follows:In Chapter2, we have proposed a simple and label-free fluorescent biosensor forthe detection of biotin based on the magnetic beads and the utilizing a SYBR Green I(SG) assisted fluorescence amplification method. SYBR Green I shows a considerablefluorescence intensity enhancement upon binding to double-stranded DNA thansingle-stranded DNA. In the absences of free biotin, biotinylaed dsDNA can be boundto the surfaces of the avidin magnetic beads at the binding sites. After embedding thefluorescent dyes of SG, a significant enhancement of the fluorescence intensity will bedetected. In the presence of free biotin, the binding sites of the avidin magnetic beadswill be occupied by the free biotin. Then the biotinylaed dsDNA can not be bound tothe surfaces of the magnetic beads. When embedding SG, there is no significantenhancement of the fluorescence intensity. Under the optimal conditions, the responseof the sensor system has a linear relationship for biotin concentration from4to24ngmL-1. The detection limit is1.19ng mL-1was calculated by the linear equation.In Chapter3, according to the work of chapter2we have build a colorimetricbiosensor for the detection of biotin based on the DNAzyme and G-quadruplex cascadefor signal amplification. In the absences of biotin, biotinylated DNAzyme will be bound to the surfaces of the magnetic beads at the binding sites. With the hairpin probe,free G-quadruplex will be released to the solution through the catalytic reaction of theDNAzyme. Then spatial structure of the G-quadruplex possess a catalytic activitywhich similar to horseradish peroxidase will be achieved when hemoglobin was addedto the solution, resulting in a color change of the ABTS solution. On the contrary, inthe presence of biotin, there will no obvious color change of the ABTS solution.Therefore, according to the color change of the solution and the signal response ofultraviolet absorption one can realize the biotin assay with rapidly and visually.In Chapter4, we introduced a single-stranded DNA (ssDNA) probe as thesubstrate of micrococcus enzyme (MNase) and the template for the silver nanocluster(DNA-AgNCs) formation simultaneously to build a simple and label-free fluorescencebiosensor for the detection of MNase activity. In the presence of MNase, ssDNA probewill be disintegrate by MNase which will inhibit the DNA-AgNCs formation, resultingin a reduce of the fluorescence intensity. In the absence of MNase, the silvernanoclusters will be formed through the ssDNA template, resulting in a increase of thefluorescence intensity. According to the fluorescence response of the sensor system,the detection of MNase will be realized. Under the optimal conditions, thefluorescence response of the sensor method showed a linear relationship for theconcentration of MNase from0to2×10-4U mL-1, a detection limit of8×10-6U mL-1was calculated through the linear equation. Some advantages of the sensor methodwere as follows: high sensitivity, good selectivity, simple operation, low cost, highflux, no need marking and not require further modification and complex design for theoligonucleotide probe. In addition, this method can also be extended to detect othernucleic acid enzymes.