Optical And Electrochemical Sensing And Detection Of Protein Recognition At Interface

Proteins play a very important role in life processes of cells and organisms.Many proteins are disease biomarkers and therapeutic targets,so it is important to prepare new biosensors of protein recognition and detection for early disease diagnosis.Optical detection of target proteins of specific recognition is readily realized based on the localized surface plasmon resonance(LSPR)effect of noble metallic nanoparticles.Surface-enhanced Raman scattering(SERS)spectroscopy,based on the LSPR effect of noble metallic nanoparticles,has been widely used for various interfacial and biological detections as a highly sensitive analytical technique.Currently,protein SERS detection is still an important and difficult topics.Molecular imprinting is to prepare synthetic polymers with the ability to specifically recognize and bind target molecules and is usually described as a technology for preparation of artificial 'lock' and artificial 'antibody'.Imprinting of proteins represents one of the most challenging tasks.There are several unsolved problems related to reduced mass transfer and permanent retention of protein templates in polymer matrices and restricted selection of aqueous media for protein imprinting.This thesis reports optical detection of myoglobin(Mb)using functionalized gold nanoparticles(AuNPs),preparation of Ag@SiO2 core/shell nanostructures and immunoassays of SERS and surface-enhanced fluorescence(SEF),and creation of protein-imprinted self-assembled monolayers(SAMs)and electrochemical detection of proteins.(1)A facile method for optical detection of Mb has been developed based on LSPR coupling of AuNPs,which is of significant importance for early disease diagnosis.Two thiol molecules containing an iminodiacetic acid moiety(IDA)were synthesized.This detection was based on the Mb-induced aggregation of IDA-functionalized AuNPs resulting from the structures of Mb sandwiched between the functionalized AuNPs via Cu2+ bridges in the coordination interactions of IDA-Cu2+-histidine residues available on the Mb surface,concomitant with a change in solution color from red to purple.The qualitative and quantitative detections of Mb were achieved by colorimetric observations and UV-vis spectral measurements,respectively.The selectivity of histidine-rich proteins assay with the functionalized AuNPs was further investigated,and it was found that the optical sensing of histidine-rich proteins was closely related to number and distribution of surface histidine residues as well as size of proteins.(2)Bifunctional nanostructured ensembles of quantum dot-decorated Ag@SiO2 nanoparticles embedded with Raman reporters were intentionally prepared for highly sensitive immunoassays by combination of SERS and SEF techniques based on the LSPR effect of noble metallic nanoparticles.The maximum SEF intensity was optimized with a silica shell spacer of about 9 nm.The SERS sensitivity was significantly improved due to the large Raman scattering cross-section of the coupling reaction product of p-aminothiophenol(PATP),generated on the Ag cores upon irradiation of laser during the SERS measurements compensated for the loss of SERS intensity resulting from silica shell coating to a great degree.Bifunctional nanostructured ensembles were used for antigen immunoassays using SERS and SEF with high sensitivity and have promising applications in bimodal bioimaging.The fluorescent carbon nanodot-decorated Ag@SiO2 nanocomposites were further prepared for fluorescence and SERS immunoassays.(3)Imprinted monolayers have several advantages over bulk imprinted polymers such as excellent mass transfer of molecules into and out of imprinted sites and transduction of binding signals detected in real time.A novel disulfide compounds containing an oligoethylene glycol(OEG)terminal moiety and two amide groups incorporated in the chain(DHAP)was synthesized.Protein-imprinted self-assembled monolayers(SAMs)were created with multiple binding sites and biocompatible imprinted cavities from template proteins,functional thiols,and DHAP through hydrogen bond and electrostatic interaction in water-acetic acid solution.DHAP played an important role in the formation of multiple binding sites and biocompatible cavities as well as resisting nonspecific protein binding.The created protein-imprinted SAMs exhibited the excellent ability of specific binding of target proteins determined by multiple binding sites and imprinted cavities.This strategy opens the possibility of controlled assembly of intellectual biomaterials and preparation of biosensors.Glycoprotein-imprinted SAMs were further created based on the reversible covalent interactions between boronic acids and cis-diols.In order to obtain boronic acid esters under neutral condition,the interactions between lactroferrin and SAMs formed from 4-mercaptophenylboronic acid(PMBA)and different amino thiols were systematically studied.The molecular length of amino thiols was required to match with that of PMBA in the SAMs for glycoprotein binding under neutral condition.Glycoprotein-imprinted SAMs were created with PMBA,PATP,and DHAP in water-ethanol solution by stepwise assembly and coassembly.It was further confirmed that the novel disulfide molecule and coassembly strategy are of universal significance in creation of protein-imprinted SAMs with specific protein binding.