Study On The Construction Of Nanomaterials Functionalized Sensing Platform For Bioanalysis

Study on the interaction of antigen and antibody, enzyme and substrate,carbonhydrate and specific protein, and detection of the biologic molecules that affectthe function of cell are the important research themes in the bioanalysis field. Theseresearches provide useful information for people to obtain the useful chemical andbiological information related to the biologic process, diagnose disease and studyon the pathogenesis. Biosensor is a powerful tool for bioanalysis. However, peoplehave to design and develop biosensor with more stability, higher sensitivity andmore selectivity because that the biological samples usually are complex and unstable.Due to the unique structure, nanomaterials have excellent physical and chemicalproperties, and they are the perfect materials for developing the sensing interface.By employing nanomaterials to construct the sensing interface, the performance ofthe biosensor including sensitivity, stability and selectivity would be greatly enhanced.The nanomaterials-based biosensors have become the hot research in the field ofbiosensor. In this dissertation, magnetic nanoparticles, gold nanoparticles, grapheneoxide and their composites were used to construct electrochemical biosensor andSPR biosensors for sensitive and selective detection of proteins and DNA. The maincontents are summarized as follows:1. Surface molecularly imprinted magnetic nanoparticles were prepared byemploying dopamine (DA) as functional monomer, hemoglobin (Hb) as template,chloroplatinic acid (H2PtCl6) as an oxidant which triggered DA polymerization onthe surface of the Fe3O4NPs. The Hb and the formed platinum nanoparticles (PtNPs) were embedded in the polydopamine (PDA). After removal of the Hb, thesurface molecularly imprinted magnetic nanoparticles (imprinted Fe3O4/PDA-Pt NPs)were formed, and then simply immobilized on the magnetic glassy carbon electrode(imprinted Fe3O4/PDA-Pt NPs/MGCE) for Hb sensing. The imprinted Fe3O4/PDA-PtNPs were demonstrated magnetic spherical shape with good solubility in water andnarrow size distribution. The Pt NPs embedded in the imprinted coating couldeffectively promote the conductivity and the mechanical strength of imprinted cavity. In an addition, the imprinted Fe3O4/PDA-Pt NPs/MGCE could selectively recognizeHb and was used to detect Hb in the range of0.14~2.7μg/mL with a detectionlimit of0.05μg/mL (S/N=3).2. By using concanavalin A (ConA) as a model protein, a novel surface plasmonresonance (SPR) sensor was developed for sensitive detection ConA. In this sensingplatform, dextran (Dex) capped gold nanoparticles (Dex-Au NPs) were initiallysynthesized in one-pot and utilized as amplification reagent. After deposition ofgraphene oxide (GO) on the SPR gold film, phenoxy-derivatized dextran (DexP)was assembled onto the GO-modified gold chip surface through π-π interaction. Theresultant GO/DexP sensing interface could specifically capture ConA which couldfurther react with Dex-Au NPs through the specific interaction between ConA andDex, forming a sandwich configuration. The morphologies and the electrochemistryof the formed sensing surface were investigated by using scanning electronmicroscopy and electrochemical techniques including electrochemical impedancespectroscopy and cyclic voltammogram. Owing to the high surface area of GO andthe excellent amplification of Dex-Au NPs, the developed sandwich SPR sensorsuccessfully fulfilled the sensitive detection of ConA in the range of1.0~20.0μg/mL with a detection limit of0.39μg/mL. Compared to the direct assay format,the prepared sandwich SPR sensor led to an improvement of28.7-fold in thesensitivity. The results demonstrated that the proposed method might provide a newdirection in designing high-performance SPR biosensors for sensitive and selectivedetection of a wide spectrum of biomolecules.3. A sensitive protocol for SPR detection of virus genes of high pathogenicbird flu H5N1was designed based on DNA multiple amplification including DNArecycle aided by lambda exonuclease (λ-exo) and hybridization chain reaction (HCR),and in-situ formed polyaniline (PANI) to enhance the SPR signals. The greatenhancement of sensitivity was attributed to three aspects: Firstly, target triggeredHCR to yield long nicked DNA double-strands with many G-quadruplex which wereable to bind with hemin and acted as horseradish peroxidase (HRP)-mimickingDNAzyme. Sencondly, G4/Hemin HRP-mimicking DNAzyme catalyzed the reactionof H2O2and aniline allowing formation polyaniline on the nicked DNA-strands, leading to great enhancement of the SPR response. Thirdly, the captured DNA (CP)with a thiol group at its3terminus and a phosphate group at its5terminus wasassembled on the gold surface. The target DNA (H5N1) could be repeatedly usedwhen it hybridized with CP because λ-exo could selectively hydrolyze the CP inthe formed dsDNA and intact H5N1is released. The detection limit of this methodwas0.27pM (S/N=3), which lowered3orders compared with the method basedon HCR DNA amplification and in-situ formed PANI to enhance the SPR response.The method also showed good differentiation between single-base mismatch DNAand completely complementary DNA. With the advantages of high sensitivity, goodselectivity and great versatility, the proposed method would have a promising futurefor detection DNA, protein and small biological molecule.4. A simple SPR sensing platform PDDA-GO/Au was developed by assemblingthe PDDA functionalized graphene oxide (PDDA-GO) on the gold surface of SPRthrough electrostatic interaction. The dsDNA with negative charges could effectivelybind to the PDDA-GO leading to the increase of negative charges of the SPRinterface and the subsequent repel of the dextran functionalized gold particles(Dex-Au NPs). On the other hand, the dsDNA could be damaged through Fentonreaction catalyzed by Cu2+, resulting in the decrease of negative charges on thesensing interface. Then, the Dex-Au NPs could be easily bound to the SPR interfaceleading to great enhancement of the SPR response. The strong interaction betweenpyrophosphate (PPi) and Cu2+suppressed the damage of dsDNA. However, alkalinephosphatase (ALP) could hydrolyze PPi to release Cu2+and thus recovery of thedamage of dsDNA, which resulted in the decrese of negative charges of sensinginterface and the bingding of Dex-Au NPs, leading to great shift of the SPR angle.The proposed method could simply, effectively, sensitively and free labeled detectALP with the detection limit of0.069U/L. Moreover, the developed method withgood selectivity could detect ALP in serum sample.