The Study On Analytic Methods Based On Novel Magnetic Protein-imprinted Materials For Early Screening Of Serious Diseases

This paper built a series of protein-imprinted materials simulating biologic probes. Based onthis, it developed multiple kinds of low-cost, simple, highly sensitive and specificelectrochemiluminescence (ECL) immunoassays by combining sandwich immunoassays andachieved highly sensitive trace assay of multiple analytes (hemoglobin, HIV-1antibody, carcinoembryonie antigen CEA and carbohydrate antigen CA199) correlating serious diseases. The detailsare as follow. The construction of hemoglobin assay is based on the fact that magnetic molecularlyimprinted polymer was used to mimick the hemoglobin antibody and could realize the enrichmentand separation of template protein after removing template protein, and achieve ultra-sensitivedetection of hemoglobin by the prepared single antibody sandwich ECL immunosensor and couldbe potentially used in early screening of cardiovascular disease. The assay method of HIV-1antibody (anti-HIV-1) is based on the epitope imprinting technology, which synthesized magneticmolecularly imprinted polymers (MMIPs) using human immunoglobulin G (HIgG) which exhibitsthe same Fc region but different Fab region to anti-HIV-1and could realize the enrichment andseparation of trace anti-HIV-1after removing template protein, then bind with antigen conjugatedwith horseradish peroxidase (HRP-HIV-1) as labels by sandwich immune reaction, and theresulting immunocomplex could catalyze the ECL of luminol-H2O2system. The obtained ECLimmunosensor could be potentially applied in early screening of AIDs. The near-simultaneousassay of CEA and CA199is based on spatial-resolved technique, which electro-polymerizedpolydopamine film as MIPs on two working electrode surface, and realized near-simultaneousassay of two tumor markers by the fabricated ECL immunosensor, which could be potentially usedin early screening of cancer. The aboved analytic methods largely lower the experiment cost,simplify the experimental steps, improve the detection sensitivity, solve the problem caused by thelimited specificity of protein-imprinted materials and provide technical support for the earlyscreening of serious diseases from clinical medicine.The study of the thesis is performed as the following fields:1. A single antibody sandwich electrochemiluminescence immunosensor based on proteinmagnetic molecularly imprinted polymers mimicking capture probes. Based on a strategy about molecular imprinting-antibody labeled probe sandwich reaction, asingle antibody sandwich ECL immunosensor for ultratrace detection of protein was developedusing MMIPs as an alternative to the first antibody as capture probes and antibodies labeled withRu-silica (Ru(bpy)32+-doped silica) doped Au (Ru@SiO2@Au) nanocomposites as labels. UsingMMIPs as capture probes will aid mass production, greatly reduce costs and improve the stabilityof immunosensor. Moreover, magnetic separation will greatly simplify the preparation and allowregeneration of the sensor. Lastly, MMIPs can enrich ultratrace levels of antigens from samplesolutions, thus providing an ultrasensitive assay. The obtained linear range was from0.1to4×104pg mL1with a detection limit of0.023pg mL1. This approach offers obvious advantages of lowcost, high sensitivity and enhanced stability compared with other immunosensors, and thereforeoffers significant potential for protein detection in a clinical laboratory setting.2. A cost-effective electrochemiluminescence sandwich immunosensor for ultrasensitivedetection of HIV-1antibody using magnetic molecularly imprinted polymers as captureprobes.In this report, a rapid and cost-effective sandwich ECL immunosensor was constructed for theultrasensitive detection of human immunodeficiency virus type1antibody (anti-HIV-1) usingMMIPs as capture probes by epitope imprinting techniques and antigen conjugated withhorseradish peroxidase (HRP-HIV-1) as labels. First,3-aminobenzeneboronic acid (APBA) wasused as the functional monomer and cross-linking reagent, which was polymerized on the surfaceof silicate-coated magnetic iron oxide nanoparticles (Fe3O4@SiO2NPs) in the presence of humanimmunoglobulin G (HIgG), as the template exhibiting the same Fc region but different Fab regionto anti-HIV-1after the addition of the initiator, ammonium persulfate. This process resulted ingrafting a hydrophilic molecularly imprinted polymer (MIP) film on the Fe3O4@SiO2NPs. Thus,MMIPs, which could be reused after eluting the template, were used to recognize and enrich ultra-trace levels of anti-HIV-1. Subsequently, a novel sandwich ECL immunosensor was formedthrough the immunoreaction between MMIPs conjugated with varied concentrations of anti-HIV-1and HRP-HIV-1. By the catalysis of HRP immobilized onto HRP-HIV-1on the ECL system ofLuminol-H2O2, a linear response range of the anti-HIV-1dilution ratio (standard positive serum)was achieved from1:20,000to1:50with a detection limit of1:60,000(S/N=3). The developedmethod provides a low-cost, simple, and sensitive way for the early diagnosis of HIV infectedpatients.3. A novel molecularly imprinted electrochemiluminescence sandwich immunosensorarray for near-simultaneous detection of CEA and CA199based on spatial-resolvedtechnique. In this study, a novel ECL sandwich immunosensor array consisting of two screen-printedcarbon working electrodes coated with molecular imprinting polymer (MIPs) to performmultiplexed immunoassay of tumor markers (carcinoembryonic antigen (CEA) and carbohydrateantigen-199(CA199)) was fabricated. Dopamine as the functional monomer of different antigens(template molecules) was electro-polymerized on the different electrode surface to formpolydopamine film as MIPs. MIPs as capture probes instead of the first antibodies and theantibodies conjugated with Ru-silica (Ru(bpy)32+-doped silica) doped poly-L-lysine (PLL) and Au(Ru@Si@PLL-Au) nanocomposites as labels were adopted. The ECL signals from the ECLimmunosensor array were near-simultaneously detected by a photomultiplier in virtue of ahomemade single-pore-two-throw switch. The obtained linear ranges for their concentration were0.05~100pg/mL for CEA and0.03~80pg/mL for CA199, with detection limits of0.02and0.01pg/mL, respectively. This novel ECL strategy based on spatial-resolved technique coupled withmolecularly imprinted technique provided a simple, low-cost, fast and sensitive approach formultiplexed immunoassay of CEA and CA199.