Surface Plasmon Resonance Sensor Based On Cyclic Amplification Assited By Enzyme

Three parts in total of work are presented in this paper:1. A new amplification method for enhancing surface plasmon resonance (SPR)signal while detecting micro-DNA is introduced. Different from traditional process ofdetection, two types of specifically designed hair-pin-shaped DNA are taken advantageto create a self-assembling chain reaction in order to improve the absorbance on thesurface of SPR device (normally gold surface). Furthermore, the application ofmagnetic beans(MB) separates the detection and amplification, with whichmicro-DNA can be regarded as signal molecule. In this experiment, ATP-aptamers arestabilized on MB. After the hybridization with conjugated micro-DNA present a signalgenerator for ATP detection. Then another ssDNA is immobilized on the surface of thegold surface for SPR. The analyte firstly reacts with the decorated MB to form signalsolution, then extract the MB and discard. The solution should be adjust to a proper pHat a certain temperature for the maximum SPR performance, then mix withamplification solution (containing hair-pin DNA for chain reaction). This mixedsolution should be injected into the SPR device. Tracing the SPR angle shift, can manget an amplified signal. The lowest detecting limit according the experimental resultreached1nM for ATP and0.5fmol/L for the signal ssDNA.Hybridization chain reaction (HCR) is another type of nucleic acid amplificationreaction and this class ofmechanisms suggests the possibility of constructingbiosensors solely from unmodified single-stranded DNA. In the present work, a SPRdetection system based on HCR was developed for amplified detection of DNA andATP. The target DNA was partly hybridized using the immobilized capture sequenceon the Au chip and the unpaired fragment of target DNA worked as a trigger to openthe hairpin DNA structures in sequence and propagate a chain reaction of hybridizationevents to self-assemble into complex structures driven by the free energy of the basepair formation without an enzyme. Amplified SPR response was observed upon theintroduction of the long nicked duplex sequence. Through specific design of the trigger sequence, the HCR based SPR sensor can be further applied for detection of ATP.2. SPR also can be used for fluorescence enhancement. Fluorescence detection inwhich SPR technology is applied has a lower detection limit than the regular one.Based on this theory, a novel multifunctional fluorescence booster was synthesized forin-cell fluorescence detection. This tiny device has a sphere-shaped (radius≥20nm)gold nano-particle as core for fluorescence enhancement. Around the metal core wasmesoporous quartz coated for a lager surface area and partially naked gold surface.This nano-ball can have three function: thiol-terminated molecule (aptamer or otherDNA for example) can attach to gold surface, quartz surface can be decorated withamino-group or carboxyl group (use APTES or APTES+succinic anhydride) and SPRfluorescence enhancement.