Studies On Fluorescent Biosensor For Adenosine/PDGF-BB Based On Applification Of Aptamer And Nanoparticles

Aptamers, obtained by the systematic evolution of ligands by exponential enrichment process, are RNA or DNA molecules with specific three-dimensional structures, compared to the commonly used antibodies, aptamers have higher affinity and specificity toward some targets, such as proteins, drugs, organic molecules and even whole cells. In addition, it also possesses some additional advantages, for example, relatively small size, lack of immunogenicity, ease of synthesis and modification. So it is attracting more and more attention in bioanalysis, chemical biology, biomedicine, and biotechnology. Nanomaterials, with their unique electronic, optical, magnetic, structural properties, the ability to be functionalized with biological recognition elements was successfully used to improve the sensitivity of bioassays, and have been widely applying in areas of sensors. Therefore it is also attracting more and more attention and application in areas of biology and chemistry. With the high selectivity and affinity of aptamers as well as the some unique properties of nanomaterials, this thesis has developed a series of fluorescence aptasensors for highly sensitive detection based on aptamers and nanomaterials. The main contents are summarized as follows:1.A method of signal enhanced fluorescence detection of adenosine based on magnetic aptamer nano-flares biosensor (MANFB) was developed. In this work, the Fe3O4/Au was used as immobilization material of thiol-aptamer,which was partly complement with a fluorescein labeled oligonucleotides (FAM-sDNA) to form aptamer nano-flares biosensor. The biosensor has no fluorescence because of the quenching effect of gold nanoparticles on AuMNPs. In the presence of adenosine, the FAM-sDNA was released from MANFB due to the combination between adenosine and its aptamer and the fluorescence was recovered after interaction of adenosine and its aptamer. In order to further enhance the fluorescent signal, ethanol was used as an enhance reagents. The enhancement intensity of fluorescence was enhanced almost three times after added ethanol in this test system. Under optimal conditions, the linear response to adenosine in the range of87μM-3.4mM and lower detection limit down to7μM (S/N=3) with the developed aptasensor.2.A new method of fluorescence resonant energy transfer for detection of adenosine using magnetic aptamer nano-flares biosensor and cationic conjugated polymer was established. In this work, The thiol-aptamer was first partly complement with a fluorescein labeled oligonucleotides (FAM-sDNA) and then immobilized on Fe3O4/Au to form magnetic aptamer nano-flares biosensor (MANFB). The biosensor has no fluorescence because of the quenching effect of gold nanoparticles on AuMNPs. In the presence of adenosine, the FAM-sDNA was released from MANFB and the fluorescence was recovered, the fluorescence resonance energy transfer (FRET) can be happened after added the cationic conjugated polymer (CCP) to the solution upon the excitation of CCP, Under optimal conditions, the linear response to adenosine in the range of 90μM-2mM and lower detection limit down to12.6μM (S/N=3) with the aptasensor.3.We also developed a fluorescence resonant energy transfer aptsensor for platelet-derived growth factor detection based on upconversion nanoparticles in30%blood serum. Upconversion fluorescent nanoparticles(UCNPs), which can convert a longer wavelength radiation (near-infrared or light infrared) into a shorter wavelength fluorescence (UV or visible) could avoid the interfering signal of biomolecules in serum. In this work, the aptamerl-Au nanoparticles (AuNPs-Aptl) and aptamer2-upconversion nanoparticles (Apt2-PUCNPs) were acted separately as acceptor and donor to demonstrate a fluorescence resonant energy transfer aptsensor. AuNPs have a good absorption properties in the visible region and Green upconversion nanoparticles have a emission at547nm upon excitation with980nm laser, in the presence of PDGF-BB, AuNPs-Aptl, PDGF-BB and Apt2-PUCNPs can fabricate the "sandwich" structure and the fluorescence resonance energy transfer (FRET) can be happened. The linear response to PDGF concentration in the range of360to1200nM with the aptsensor. This detection approach can also exhibit good stability, sensitively and higly specific, it shows promising potential in other protein monitoring and disease diagnosis.