Preparation Of Neuropeptide Molecularly Imprinted Polymer Array Chip And Its Fluorescence Immunoassay Analysis Research

Cholecystokinin and enkephalins are two kinds of neuropeptides with multifunctionality and widespread distribution.These two neuropeptides are intimately involved in a diversity of normal behaviors such as neurotransmission,epilepsy depression and pain sensing.In the majority of previous reports,these two neuropeptides were quantified by radioimmunoassay and enzyme linked immunosorbent assay.Although these immune-based techniques are highly sensitive,they require highly specific antibodies that are expensive to develop,complicated to operate and have radioactive pollution.Thus,it is of great significance to develop a economic,highly-selective artificial antibody to replace the biological antibody for analysis of protein.Molecularly imprinted polymer(MIP)biochip is the integration of MIP and biochip with selective recognition toward target molecules.The novel array chip,which combines the characters of MIP and biochip,has both the merits of MIP such as the specificity recognition,resistance to acid,alkali and high temperature and and the merits of biochip including the high throughput,high efficiency and low consumption.This approach combines the selective recognition property of MIP,high-throughput character of microarray and high sensitivity of fluorescence detection.In this work,a novel molecularly imprinted polymer array chip for highly selective recognition of encephalin,a molecularly imprinted microplate and a molecularly imprinted membrane for highly selective recognition of cholecystokinin.were prepared.Employed this molecularly imprinted polymer array and moleculayly imprinted microplate for high throughput detection of enkephalinor cholecystokinin in human cerebrospinal fluid.The major contents of this thesis are described as follows:1.A novel biomimetic array chip for enkephalins,which uses the MIP as the sensing materials was investigated.The MIP array for highly selective recognition of enkephalins was prepared on glass slides by integration of photolithography and epitope imprinting technique.Reaction parameters affecting the synthesis of MIP were optimized.The binding energy values of template-monomer complexes were calculated by molecular simulation.The synthesized MIP material was characterized by employing scanning electron microscopy and infrared spectroscopy;and its adsorption capacity and selectivity were evaluated using equilibrium adsorption tests and MALDI-TOF MS analysis.This MIP microarray was utilized as a biomimetic sensor and an imaging assay,which is analogous to solid-phase competitive immunoassay for quantification of enkephalins was developed via fluorescence detection.This MIP microarray-based competitive immunoassay was applied for the detecting of enkephalins in human cerebrospinal fluids.2.Molecularly imprinted polymer microsphere as the artificial biomimetic recognition element for cholecystokinin neuropeptides was parepared by integration of precipitation polymerization and epitope imprinting technique.And then coated in microplates with polyvinyl alcohol(PVA)as glue.Influencing factors were investigated and optimized in detail.The synthesized MIP material was characterized by employing scanning electron microscopy,and its adsorption capacity and selectivity were evaluated using equilibrium adsorption tests.Based on this moleculayly imprinted microplate biomimetic chip,an imaging assay,which is analogous to solid-phase competitive immunoassay for high throughput detection of cholecystokinin in human cerebrospinal fluid was developed via fluorescence detection.3.A molecularly imprinted polymer membrane for highly selective recognition of cholecfstokinin neuropeptides was synthesized on glass slide by epitope imprinting technique.The polymerization conditions were investigated and optimized.The synthesized MIP membrane was characterized by scanning electron microscopy and infrared spectroscopy,and its adsorption capacity and selectivity were evaluated using equilibrium adsorption tests.An imaging assay,which is analogous to solid-phase competitive immunoassay,was developed using this MIP membrane as the support for fluorescence reaction and used for quantification of CCK neuropeptides in human cerebrospinal of detection for CCK neuropeptides fluids by inverted fluorescence microscope-CCD imaging system.