Preparation And Analytical Application Of Chemiluminescence Functionalized Magnetic Microbeads And Study On Single Particle Chemiluminescence Imaging

In this dissertation,the concept and principle of chemiluminescence(CL),the application of magnetic microbeads in the field of CL and the state of arts of single particle optical imaging technology were reviewed.It was point out that CL functionalized magnetic microbeads with high CL efficiency in label-free bioassays are highly desired.In this work,the preparation,analytical application and single single particle particle CL imaging of functionalized magnetic microbeads were studied.Bifunctional magnetic microbeads were successfully synthesized by functionalizing CL reagents and metal ions on the surface of magnetic microbeads,and their CL properties were studied.A label-free CL aptasensor for 2,4,6-trinitrotoluene(TNT)was constructed by using the functionalized magnetic microbeads as carrier and CL sensing interface.A single particle CL imaging system was designed and built.And a microfluidic chip with microporous array was developed to immobilize magnetic microbeads.The CL images and kinetic curves of single magnetic microbeads were obtained successfully.The structure-activity relationship of individual particles was explored,and the CL functionalized magnetic microbeads was further optimized for improving the analytical performance of the proposed aptasensor.The main contents are as follows:1.Highly CL magnetic beads containing N-(4-aminobutyl)-N-ethyl isoluminol(ABEI)and Co2+(Co2+/ABEI/MBs)were first synthesized via a facile strategy.ABEI first reacted with carboxylated MBs by amidation reaction to prepare ABEI/MBs,which then was mixed with Co2+ to form Co2+/ABEI/MBs.The morphology and composition of Co2+/ABEI/MBs composites were characterized by scanning electron microscopy(SEM),X-ray photoelectron spectroscopy(XPS)and inductively coupled plasma atomic emission spectrometry(ICP-AES).It was found that the carboxylated magnetic beads have an average diameter of～55 μm,and the as-prepared spherical Co2+/ABEI/MBs showed excellent monodispersion.Moreover,the assembly mechanism of Co2+/ABEI/MBs composites has been proposed as follows.First,ABEI was immobilized on the surface of carboxylated MBs via a typical EDC/NHS coupling reaction.And then,positively charged Co2+ could be easily adsorbed on the surface of MBs through electrostatic interaction due to the carboxyl full-covered MBs were negatively charged around pH 7.0.The as-preparedCo2+/ABEI/MBs exhibited good paramagnetic properties,satisfactory stability,and intense CL emission when reacted with H2O2,which was more than 150 times that of ABEI functionalized MBs.It is suggested that heterogeneous catalysis of Co2+immobilized on the MBs facilitated the formation of HO· radicals,which reacted with ABEI also immobilized on the surface of MBs and the dissolved O2 to yield ABEI·-and O2·-.Finally,ABEI·-reacted with O2·-to produce strong CL emission.Both immobilization of Co2+ and ABEI highly concentrated Co2+ and ABEI molecules on the surface of MBs and the surface of MBs stimulated electron transfer,which also contributed to strong light emission.The as-prepared CL functionalized magnetic microbeads have excellent CL properties and good para-magnetism,which enable them to have the capability of magnetic separation and lay a foundation for further application in bioassays.2.Based on Co2+/ABEI/MBs,a label-free aptasensor was firstly developed for sensitive detection of 2,4,6-Trinitrotoluene(TNT).It was found that TNT aptamer could attach to the surface of Co2+/ABEI/MBs via electrostatic interaction and coordination interaction between TNT aptamer and Co2+,leading to a decrease in CL intensity due to that catalytic site Co2+ was blocked by the aptamer.In the presence of TNT,TNT would bind strongly with TNT aptamer and detach from the surface of Co2+/ABEI/MBs,resulting in partial restoration of the CL signal.Accordingly,label-free aptasensor was developed for the determination of TNT in the range of 0.05-25 ng/mL with a detection limit of 17 pg/mL.This work demonstrates that Co2+/ABEI/MBs is easily connected with recognition biomolecules,which is not only a magnetic carrier but also a direct sensing interface with excellent CL activity.It provides a novel CL interface with magnetic property which is easily to separate analytes from sample matrix to construct label-free bioassays.3.A single particle CL imaging system has been successfully designed and built.The system consists of an Olympus BX53 microscope,a high performance EMCCD(Andor,iXon-Ultra-897),an optical platform equipped with isolation system and a computer equipped with EMCCD control software and data analysis software.Devices,optical path and software operating system were optimized.In order to firmly trap and immobilize the microbeads on a slide,the immobilization method of magnetic microbeads was studied.A polydimethylsiloxane(PDMS)apparatus contained an array of tens of microwells with varying diameter was designed and fabricated,so that the single microbeads can keep their original location without moving during chemical reactions.This was not only necessary for extracting the CL kinetics with image analysis,but also particularly important for characterizing the very same microbeads after the reaction.The single particle CL image was successfully obtained.This system lays a foundation for the subsequent study of single particle CL imaging.4.The built optical microscope in the above was used to acquire the CL emission from single magnetic polymer hybrid microbeads functionalized with luminol analogues,and to access the CL kinetics of each individual particle.It was incidentally found that a minor subpopulation of microbeads exhibited intense and delayed CL emission while the majority showed transient and weak emission.Structural characterization of the very same individual particles uncovered that the amorphous multicore microstructures were responsible for the enhanced encapsulation efficiency and optimized CL reaction kinetics.Guided by this knowledge stemming from single particle CL imaging,the synthesis procedure was rationally optimized to enrich the portion of microbeads with better CL performance,which was validated by both single particle imaging and the significantly improved analytical performance at the ensemble level.The present work not only demonstrates the CL imaging and CL kinetics curve of single microbeads for the first time,but also sets a clear example showing the capability of single particle studies to investigate the structure-activity relationship in a bottom-up manner and to help the rational design of ensemble materials with improved performance.