Ag @ SiO 2 Core-shell Nanoparticles Enhanced Fluorescence Analysis

Metal-Enhanced Fluorescence (MEF) is a phenomenon which the fluorescence emission intensity of fluorophores has a substantial increase compared to that of the free dyes, resulted from that the fluorophores were fixed to the surface of metal or metal nanoparticles with the appropriate distance. MEF has incomparable use in the field of fluorescence resonance energy transfer immunoassay, DNA sequencing, single molecule fluorescence detection and fluorescent probe.Because of the special properties of localized surface plasmon resonance (LSPR), metal nanomaterials have good application prospects in the field of biology, medicine and materials science. Particularly, silver nanomaterials have received more and more attention due to their remarkable enhancement effect of localized electromagnetic field, larger scattering areas and high refraction sensitivity. However, there are several shortcomings, such as poor stability and easy to agglomerate, which seriously limit the practical applications of silver nanoparticles (AgNPs). AgNPs can be surface-modified with core-shell structures to improve their stability and modulate the distance between the AgNPs and fluorophores to optimize the fluorescence efficiency, which have an important significance on the promotion of their practical applications. The silica not only has high stability and good biocompatibility, but also its surface is easy to be chemically modified making it an excellent material for shaping the shell.Based on the distance modulation effect in MEF, in this thesis, AgNPs were coated with the silica shell thickness of ca.8nm. By controlling the distance between the fluorescent molecules and AgNPs using the silica, the MEF has been achieved. Then, the new method for detection of adenosine triphosphate (ATP) and assay of acetylcholinesterase (AChE) activity have been developed.The main contents of this thesis as follows:The first chapter is the introduction which gives a brief overview of the mechanism and current research status of the MEF, as well as the advantages, preparation and applications of metal core-shell nanomaterials.In the second chapter, based on the enhanced fluorescence phenomenon of Ag@SiO2core-shell nanoparticles, a new method to determine ATP was developed. At first, AgNPs were prepared with average diameter at about50±3nm via ascorbic acid reduction method. Then, using classical Stober method, in alcohol phase, we synthesized Ag@SiO2with the silica shell thickness of8±1nm by controlling the concentration of tetraethoxysilane (TEOS). A complementary DNA (cDNA) probe modified with-NH2at5’end was then immobilized onto the surface of the silica shell and hybridized with the ATP-binding aptamer labeled with Cy5at the3’end (aptamer-Cy5). So that the distance between AgNPs and Cy5is modulated to ca.8nm. At this time, the32-fold fluorescence enhancement can be detected. When adding the target molecules ATP, the aptamer binded to ATP due to their high affinity and dissociated from the surface of Ag@SiO2.Thus the distance between Cy5and AgNPs became uncontrollable and the fluorescence decreased. This "on-off" format made this approach sensitive and effective. Detection of ATP has a liner response from0-0.5mM with a detection limit of8μM. Moreover, we measured the concentration of ATP in the blood samples and the recoveries were determined to be89-94%, which confirmed the validity of this assay.In chapter three, based on the enhanced fluorescence phenomenon of Ag@SiO2core-shell nanoparticles, we developed a new platform to determine AChE and screen AChE inhibitors. The detection principle is mainly involved in catalytic hydrolysis of AChE and electrostatic adsorption. At first, AChE catalyzed the hydrolysis of the substrate acetylthiocholine chloride (ATChCl) and generated positively charged thiocholine (TCh) which can be adsorbed to the negatively charged surface of Ag@SiO2via electrostatic interaction. Then, at pH value of7.5, fluorescent dye,8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) is negatively charged which can be adsorbed onto the positively charged surface of Ag@SiO2with TCh, resulting in the MEF effect. Therefore, the AChE activity can be determined and its inhibitors can be screened. A low detection limit of0.00005U/mL AChE was obtained. We studied tacrine and isofraxidin as model inhibitors. The results showed that they both can inhibit the activity of AChE. The IC5value of tacrine is9.9±0.4nM and that of isofraxidin is16.5±0.6nM. Therefore, tacrine has better inhibition effect on AChE.